Methods For Directed Folding Assembly Or Dimerization Of Proteins By Templated Assembly Reactions

ABSTRACT

The present disclosure provides nucleic acid molecules, compositions, and kits comprising the same, and methods for producing templated assembly products.

FIELD

The present disclosure is directed, in part, to nucleic acid molecules,compositions, and kits comprising the same, and methods for producingtemplated assembly products.

BACKGROUND

A goal of drug development is delivering potent bio-therapeuticinterventions to pathogenic cells, such as virus infected cells,neoplastic cells, cells producing an autoimmune response, and otherdysregulated or dysfunctional cells. Examples of potent bio-therapeuticinterventions capable of combating pathogenic cells include toxins,pro-apoptotic agents, and immunotherapy approaches that re-direct immunecells to eliminate pathogenic cells. Unfortunately, developing theseagents is extremely difficult because of the high risk of toxicity toadjacent normal cells or the overall health of the patient.

A method that has emerged to allow delivery of potent interventions topathogenic cells while mitigating toxicity to normal cells is targetingof therapeutics by directing them against molecular markers specific forpathogenic cells. Targeted therapeutics have shown extraordinaryclinical results in restricted cases, but are currently limited in theirapplicability due to a lack of accessible markers for targeted therapy.It is extremely difficult, and often impossible, to discover proteinmarkers for many pathogenic cell types.

More recently, therapies targeted to nucleic acid targets specific topathogenic cells have been developed. Existing nucleic acid-targetedtherapies, such as siRNA, are able to down-modulate expression ofpotentially dangerous genes, but do not deliver potent cytotoxic orcytostatic interventions and thus are not particularly efficient ateliminating the dangerous cells themselves.

Hence, there exists a need to combat the poor efficacy and/or severeside effects of existing bio-therapeutic interventions. As describedherein, proteins can be assembled via folding or dimerization usingnucleic acid molecule templates which can be used to combat pathogenicor otherwise undesirable cells or cell products. Such templated assemblyprocesses can be used to target the cell types of interest fordestruction. Pairs of modified oligonucleotides carrying speciallytailored and mutually reactive ligands can assemble proteins withpredetermined functions following templated assembly as set forthherein.

SUMMARY

The present disclosure provides haplomer-ligand complexes comprising: ahaplomer, wherein the haplomer comprises a polynucleotide that issubstantially complementary to a target nucleic acid molecule; and aligand linked to the 5′ or 3′ terminus of the haplomer, wherein theligand comprises a ligand partner binding site.

The present disclosure also provides compositions or kits comprising afirst haplomer-ligand complex described herein and a secondhaplomer-ligand complex described herein, wherein: the ligand of thefirst haplomer-ligand complex is linked to the 5′ terminus of thepolynucleotide of the first haplomer-ligand complex: and the ligand ofthe second haplomer-ligand complex is linked to the 3′ terminus of thepolynucleotide of the second haplomer-ligand complex.

The present disclosure also provides bottle haplomer-ligand complexescomprising: a bottle haplomer, wherein the bottle haplomer comprises apolynucleotide, wherein the polynucleotide comprises: a) a first stemportion comprising from about 10 to about 20 nucleotide bases; b) ananti-target loop portion comprising from about 16 to about 40 nucleotidebases and having a first end to which the first stem portion is linked,wherein the anti-target loop portion is substantially complementary to atarget nucleic acid molecule; c) a second stem portion comprising fromabout 10 to about 20 nucleotide bases linked to a second end of theanti-target loop portion, wherein the first stem portion issubstantially complementary to the second stem portion; and d) a ligandlinked to the terminal end of either the first stem portion or thesecond stem portion, wherein the ligand comprises a ligand partnerbinding site; wherein the T_(m) of the anti-target loop portion:targetnucleic acid molecule is greater than the T_(m) of the first stemportion:second stem portion.

The present disclosure also provides compositions or kits comprising: abottle haplomer-ligand complex described herein; and a secondhaplomer-ligand complex comprising: a nucleotide portion comprising fromabout 6 to about 20 nucleotide bases that is substantially complementaryto the stem portion of the bottle haplomer-ligand complex that is linkedto the ligand of the bottle haplomer-ligand complex; and a ligand linkedto the 5′ or 3′ terminus of the nucleotide portion of the secondhaplomer-ligand complex, wherein the ligand comprises a ligand partnerbinding site; wherein the T_(m) of the second haplomer-ligandcomplex:first or second stem portion linked to the ligand of the bottlehaplomer-ligand complex is less than or equal to the T_(m) of the firststem portion:second stem portion of the bottle haplomer-ligand complex.

The present disclosure also provides compounds having the formula:

where m is from 3 to 6.

The present disclosure also provides compounds having the formula:

where m is from 3 to 6.

The present disclosure also provides compounds having the formula:

where n is from 1 to 6.

The present disclosure also provides compounds having the formula:

where x is from 1 to 6.

The present disclosure also provides compounds having the formula:

where x is from 1 to 6.

The present disclosure also provides compounds having the formula:

which is also referred to herein as Monovalent FKBP Ligand-2 (MFL2).

The present disclosure also provides fusion proteins comprising afragment of a protein of interest fused to a ligand binding domain,wherein: the ligand binding domain is a ligand binding domain for smallmolecule ligands; or the ligand binding domain is an interactive proteindomain.

The present disclosure also provides compositions or kits comprising afirst fusion protein described herein and a second fusion proteindescribed herein, wherein the protein of interest of the first fusionprotein and the protein of interest of the second fusion protein candimerize or fold together.

The present disclosure also provides compositions or kits comprising: afirst haplomer-ligand complex described herein; a second haplomer-ligandcomplex described herein; a first fusion protein described herein; and asecond fusion protein described herein; wherein the ligand of the firsthaplomer-ligand complex is linked to the 5′ terminus of thepolynucleotide of the first haplomer-ligand complex; wherein the ligandof the second haplomer-ligand complex is linked to the 3′ terminus ofthe polynucleotide of the second haplomer-ligand complex; wherein thepolynucleotide of the first haplomer-ligand complex is substantiallycomplementary to a target nucleic acid molecule; wherein thepolynucleotide of the second haplomer-ligand complex is substantiallycomplementary to the target nucleic acid molecule at a site in spatialproximity to the polynucleotide of the first haplomer-ligand complex;wherein the ligand of the first haplomer-ligand complex and the ligandbinding domain of the first fusion protein can interact; wherein theligand of the second haplomer-ligand complex and the ligand bindingdomain of the second fusion protein can interact; and wherein thefragment of the protein of interest of the first fusion protein and thefragment of the protein of interest of the second fusion protein candimerize or fold together.

The present disclosure also provides compositions or kits comprising: afirst haplomer-ligand complex described herein; a second haplomer-ligandcomplex described herein; a first fusion protein described herein; and asecond fusion protein described herein; wherein the ligand of the firsthaplomer-ligand complex is linked to the 5′ terminus of thepolynucleotide of the first haplomer-ligand complex; wherein the ligandof the second haplomer-ligand complex is linked to the 3′ terminus ofthe polynucleotide of the second haplomer-ligand complex; wherein thepolynucleotide of the first haplomer-ligand complex is substantiallycomplementary to a target nucleic acid molecule; wherein thepolynucleotide of the second haplomer-ligand complex is substantiallycomplementary to the target nucleic acid molecule at a site in spatialproximity to the polynucleotide of the first haplomer-ligand complex;wherein the ligand of the first haplomer-ligand complex and the ligandbinding domain of the first fusion protein can interact; wherein theligand of the second haplomer-ligand complex and the ligand bindingdomain of the second fusion protein can interact; and wherein thefragment of the protein of interest of the first fusion protein and thefragment of the protein of interest of the second fusion protein candimerize or fold together.

The present disclosure also provides compositions or kits comprising: afirst bottle haplomer-ligand complex described herein; a secondhaplomer-ligand complex described herein, wherein the secondhaplomer-ligand complex comprises a nucleotide portion that issubstantially complementary to the stem portion of the bottlehaplomer-ligand complex that is linked to the ligand of the bottlehaplomer-ligand complex; a first fusion protein described herein; and asecond fusion protein described herein; wherein the ligand of the firstbottle haplomer-ligand complex and the ligand binding domain of thefirst fusion protein can interact; wherein the ligand of the secondhaplomer-ligand complex and the ligand binding domain of the secondfusion protein can interact; and wherein the fragment of the protein ofinterest of the first fusion protein and the fragment of the protein ofinterest of the second fusion protein can dimerize or fold together.

The present disclosure also provides compositions or kits comprising: afirst bottle haplomer-ligand complex described herein; a secondhaplomer-ligand complex described herein, wherein the secondhaplomer-ligand complex comprises a nucleotide portion that issubstantially complementary to the stem portion of the bottlehaplomer-ligand complex that is linked to the ligand of the bottlehaplomer-ligand complex; a first fusion protein described herein; and asecond fusion protein described herein; wherein the ligand of the firstbottle haplomer-ligand complex and the ligand binding domain of thefirst fusion protein can interact; wherein the ligand of the secondhaplomer-ligand complex and the ligand binding domain of the secondfusion protein can interact; and wherein the fragment of the protein ofinterest of the first fusion protein and the fragment of the protein ofinterest of the second fusion protein can dimerize or fold together.

The present disclosure also provides methods for the directed assemblyof a protein comprising: contacting a target nucleic acid molecule witha first haplomer-ligand complex described herein; contacting the targetnucleic acid with a second haplomer-ligand complex described herein;contacting the first haplomer-ligand complex with a first fusion proteindescribed herein; and contacting the second haplomer-ligand complex witha second fusion protein described herein; wherein the ligand of thefirst haplomer-ligand complex is linked to the 5′ terminus of thepolynucleotide of the first haplomer-ligand complex; wherein the ligandof the second haplomer-ligand complex is linked to the 3′ terminus ofthe polynucleotide of the second haplomer-ligand complex; wherein thepolynucleotide of the first haplomer-ligand complex is substantiallycomplementary to a target nucleic acid molecule; wherein thepolynucleotide of the second haplomer-ligand complex is substantiallycomplementary to the target nucleic acid molecule at a site in spatialproximity to the polynucleotide of the first haplomer-ligand complex;wherein the ligand of the first haplomer-ligand complex and the ligandbinding domain of the first fusion protein can interact; and wherein theligand of the second haplomer-ligand complex and the ligand bindingdomain of the second fusion protein can interact; thereby resulting inthe folding or dimerization of the fragment of the protein of interestof the first fusion protein with the fragment of the protein of interestof the second fusion protein.

The present disclosure also provides methods for the directed assemblyof a protein comprising: contacting a target nucleic acid molecule witha first haplomer-ligand complex described herein; contacting the targetnucleic acid with a second haplomer-ligand complex described herein;contacting the first haplomer-ligand complex with a first fusion proteindescribed herein; and contacting the second haplomer-ligand complex witha second fusion protein described herein; wherein the ligand of thefirst haplomer-ligand complex is linked to the 5′ terminus of thepolynucleotide of the first haplomer-ligand complex; wherein the ligandof the second haplomer-ligand complex is linked to the 3′ terminus ofthe polynucleotide of the second haplomer-ligand complex; wherein thepolynucleotide of the first haplomer-ligand complex is substantiallycomplementary to a target nucleic acid molecule; wherein thepolynucleotide of the second haplomer-ligand complex is substantiallycomplementary to the target nucleic acid molecule at a site in spatialproximity to the polynucleotide of the first haplomer-ligand complex;wherein the ligand of the first haplomer-ligand complex and the ligandbinding domain of the first fusion protein can interact; and wherein theligand of the second haplomer-ligand complex and the ligand bindingdomain of the second fusion protein can interact; thereby resulting inthe folding or dimerization of the fragment of the protein of interestof the first fusion protein with the fragment of the protein of interestof the second fusion protein.

The present disclosure also provides methods for the directed assemblyof a protein comprising: contacting a target nucleic acid molecule witha complex formed by the interaction of a first haplomer-ligand complexdescribed herein with a first fusion protein described herein, whereinthe ligand of the first haplomer-ligand complex is linked, to the 5′terminus of the polynucleotide of the first haplomer-ligand complex, andwherein the ligand of the first haplomer-ligand complex interacts withthe ligand binding domain of the first fusion protein; and contactingthe target nucleic acid molecule with a complex formed by theinteraction of a second haplomer-ligand complex described herein with asecond fusion protein described herein, wherein the ligand of the secondhaplomer-ligand complex is linked to the 5′ terminus of thepolynucleotide of the second haplomer-ligand complex, and wherein theligand of the second haplomer-ligand complex interacts with the ligandbinding domain of the second fusion protein; thereby resulting in thefolding or dimerization of the fragment of the protein of interest ofthe first fusion protein with the fragment of the protein of interest ofthe second fusion protein.

The present disclosure also provides methods for the directed assemblyof a protein comprising: contacting a target nucleic acid molecule witha complex formed by the interaction of a first haplomer-ligand complexdescribed herein with a first fusion protein described herein, whereinthe ligand of the first haplomer-ligand complex is linked to the 5′terminus of the polynucleotide of the first haplomer-ligand complex, andwherein the ligand of the first haplomer-ligand complex interacts withthe ligand binding domain of the first fusion protein; and contactingthe target nucleic acid molecule with a complex formed by theinteraction of a second haplomer-ligand complex described herein with asecond fusion protein described herein, wherein the ligand of the secondhaplomer-ligand complex is linked to the 5′ terminus of thepolynucleotide of the second haplomer-ligand complex, and wherein theligand of the second haplomer-ligand complex interacts with the ligandbinding domain of the second fusion protein; thereby resulting in thefolding or dimerization of the fragment of the protein of interest ofthe first fusion protein with the fragment of the protein of interest ofthe second fusion protein.

The present disclosure also provides methods for the directed assemblyof a protein comprising: contacting a target nucleic acid molecule witha bottle haplomer-ligand complex described herein; contacting the targetnucleic acid with a second haplomer-ligand complex described herein,wherein the second haplomer-ligand complex comprises a nucleotideportion that is substantially complementary to the stem portion of thebottle haplomer-ligand complex that is linked to the ligand of thebottle haplomer-ligand complex; contacting the bottle haplomer-ligandcomplex with a first fusion protein described herein, wherein the ligandof the bottle haplomer-ligand complex and the ligand binding domain ofthe first fusion protein can interact; and contacting the secondhaplomer-ligand complex with a second fusion protein described herein,wherein the ligand of the second haplomer-ligand complex and the ligandbinding domain of the second fusion protein can interact; therebyresulting in the folding or dimerization of the fragment of the proteinof interest of the first fusion protein with the fragment of the proteinof interest of the second fusion protein.

The present disclosure also provides methods for the directed assemblyof a protein comprising: contacting a target nucleic acid molecule witha bottle haplomer-ligand complex described herein; contacting the targetnucleic acid with a second haplomer-ligand complex described herein,wherein the second haplomer-ligand complex comprises a nucleotideportion that is substantially complementary to the stem portion of thebottle haplomer-ligand complex that is linked to the ligand of thebottle haplomer-ligand complex; contacting the bottle haplomer-ligandcomplex with a first fusion protein described herein, wherein the ligandof the bottle haplomer-ligand complex and the ligand binding domain ofthe first fusion protein can interact; and contacting the secondhaplomer-ligand complex with a second fusion protein described hereinwherein the ligand of the second haplomer-ligand complex and the ligandbinding domain of the second fusion protein can interact; therebyresulting in the folding or dimerization of the fragment of the proteinof interest of the first fusion protein with the fragment of the proteinof interest of the second fusion protein.

The present disclosure also provides methods for the directed assemblyof a protein comprising: contacting a target nucleic acid molecule witha bottle haplomer-ligand complex described herein; contacting the targetnucleic acid molecule with a second haplomer-ligand complex describedherein, wherein the second haplomer-ligand complex comprises anucleotide portion that is substantially complementary to the stemportion of the bottle haplomer-ligand complex that is linked to theligand of the bottle haplomer-ligand complex; contacting the bottlehaplomer-ligand complex with a first fusion protein described herein,wherein the ligand of the bottle haplomer-ligand complex and the ligandbinding domain of the first fusion protein can interact; and contactingthe second haplomer-ligand complex with a second fusion proteindescribed herein, wherein the ligand of the second haplomer-ligandcomplex and the ligand binding domain of the second fusion protein caninteract; thereby resulting in the folding or dimerization of thefragment of the protein of interest of the first fusion protein with thefragment of the protein of interest of the second fusion protein.

The present disclosure also provides methods for the directed assemblyof a protein comprising: contacting a target nucleic acid molecule witha bottle haplomer-ligand complex described herein; contacting the targetnucleic acid molecule with a second haplomer-ligand complex describedherein, wherein the second haplomer-ligand complex comprises anucleotide portion that is substantially complementary to the stemportion of the bottle haplomer-ligand complex that is linked to theligand of the bottle haplomer-ligand complex; contacting the bottlehaplomer-ligand complex with a first fusion protein described herein,wherein the ligand of the bottle haplomer-ligand complex and the ligandbinding domain of the first fusion protein can interact; and contactingthe second haplomer-ligand complex with a second fusion proteindescribed herein, wherein the ligand of the second haplomer-ligandcomplex and the ligand binding domain of the second fusion protein caninteract; thereby resulting in the folding or dimerization of thefragment of the protein of interest of the first fusion protein with thefragment of the protein of interest of the second fusion protein.

The templated assembly of functionally active proteins by dimerizationor folding from protein fragments associated with modifiedoligonucleotides (haplomers) may be divided into a two-stage process.The first stage comprises the binding of haplomers to theircomplementary counterparts. The ligand-mediated second stage enableshomo- or heterodimerization of protein fusions with appropriateligand-binding domains. Template-mediated dimerization or folding isapplicable to the activation of specific proteins in target pathologicalcells, for diagnostic or therapeutic purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an overview of a representative Ligand Directed TemplateAssembly by Proximity-Enhanced Reactivity (LD-TAPER) principle.

FIG. 2 shows representative ternary complex formation between FK506,FKBP, and calcineurin; and rapamycin and mTOR/FRB and calcineurin.

FIG. 3 shows a representative scope and range of LD-TAPER.

FIG. 4 shows a representative “enforced dimerization” of proteins ofinterest expressed as fusions with FKBP or FRB domains, mediated bycompounds derived from FK506 or rapamycin.

FIG. 5 shows a representative LD-TAPER: Protein dimerization by smallmolecule mediators.

FIG. 6 shows a representative modified monovalent version of a mutantFKBP-binding compound, FKM-NHS.

FIG. 7 shows a representative modified monovalent version of a mutantFKBP-binding compound, FKM-sulfo-NHS.

FIG. 8 shows a representative modified monovalent version of a mutantFKBP-binding compound, FKM-PEG3-NHS.

FIG. 9 shows a representative small molecule-mediated proteindimerization via LD-TAPER.

FIG. 10 shows another representative small molecule-mediated proteindimerization via LD-TAPER.

FIG. 11 shows another representative small molecule-mediated proteindimerization via LD-TAPER.

FIG. 12 shows another representative small molecule-mediated proteindimerization via LD-TAPER.

FIG. 13 shows representative structures of locked TAPER oligonucleotidesfor small molecule-mediated protein dimerization via LD-TAPER, usingsmall-molecule ligands.

FIG. 14 shows a representative LD-TAPER:Protein dimerization by smallinteractive protein domains.

FIG. 15 shows representative polarity considerations for parallelleucine zippers.

FIG. 16 shows a representative. LD-TAPER:Split-Protein Folding by smallmolecule ligand/ligand binding domain interactions.

FIG. 17 shows a representative LD-TAPER:Split-Protein Folding by smallprotein domain interactions, and polarity effects.

FIG. 18 shows representative heterocomponent systems for LD-TAPER.

FIG. 19 shows representative heterocomponent systems forLD-TAPER:Specific embodiments with leucine zippers.

FIG. 20 shows a representative LD-TAPER ligand stabilization, by meansof appending click groups onto haplomeric ligands for templated in situjoining.

FIG. 21 shows a representative click-modified monovalent ligand with amethyltetrazine (MTZ) side-chain for mutant FKBP and oligonucleotidederivatization.

FIG. 22 shows a representative click-modified monovalent ligand with atrans-cyclooctene (TCO) side chain for mutant FKBP and oligonucleotidederivatization.

FIG. 23 shows a representative testing expression of c-Jun fragments asfusion proteins with Maltose-Binding Protein (MBP), and demonstration ofproduction of free c-Jun products after enterokinase cleavage of thefusion.

FIG. 24 shows a representative Locked TAPER c-Jun oligonucleotides forsplit-protein folding by LD-TAPER, using an antiparallel zipperconfiguration.

FIG. 25 shows a representative Locked TAPER split-protein folding byLD-TAPER with c-Jun:c-Fos interactions.

FIG. 26 shows a representative Locked TAPER compoundFKM-oligonucleotides for split-protein folding by LD-TAPER.

FIG. 27 shows a representative Locked TAPER split-protein folding byLD-TAPER with FKM-FKBP binding.

FIG. 28 shows expressed Gaussia split-protein FKBP and FRB fusionproteins, with and without respective C22S and C61S mutations, tested on16% Tricine gel; shown are expressed samples post-induction with IPTG,where ‘Direct Lysates’ refers to whole cell samples taken and lysed instandard Laemmli SDS buffer before gel loading, and ‘IMAC PurifiedProteins’ refers to samples after elution with imidazole fromhexahistidine affiinty-magnetic beads; lanes: 1 & 7, N-terminal Gaussiasegment-FKBP(F36V); 2 & 8, FKBP(F36V)-C-terminal Gaussia segment; 3 & 9,FRB-C-terminal Gaussia segment; 4 & 10, N-terminal Gaussiasegment-FKBP(F36V/C22S); 5 & 11, FKBP(F36V/C22S)-C-terminal Gaussiasegment; 6 & 12, FRB(C61S)-C-terminal Gaussia segment.

FIG. 29 (panels 1, 2, 3, and 4) shows dimerization responses ofsplit-protein Gaussia. fragments fused with either the FKBP-F36V domain,with or without additional C22S mutations, or the FRB domain, with orwithout additional C61S mutations; data shows luminescence responses forproteins with and without dimerizers, all at 4 hours from start of thetest; 1: Gaussia N-terminal-FKBP-F36V fusion (A), FKBP-F36V-C-terminalGaussia fusion (B), combination (A)+(B), and (A)+(B) with 0.15 μMAP20187 (AP); 2: Gaussia N-terminal-FKBP-F36V-C22S fusion (C),FKBP-F36V-C22S-C-terminal Gaussia fusion (D), combination (C)+(D), and(C)+(D) with 0.15 μM AP20187 (AP); 3: Gaussia N-terminal-FKBP-F36Vfusion (A), FRB-C-terminal Gaussia fusion (V), (A)+(V), and (A)+(V) with0.15 μM rapamycin (RAP); 4: Gaussia N-terminal-FKBP-F36V-C22S fusion(C0, FRB-C61S-C-terminal Gaussia fusion (W), (C)+(W), and (C) (W) with0.15 μM rapamycin (RAP).

FIG. 30 shows conjugate formation via reaction between 5′- or3′-thiol-modified oligonucleotides and monovalent FKBP-F36V Ligand MFL2;each thiol group on the modified oligonucleotides is separated from thenearest base by a 6C spacer; 33 pmol of unmodifiedthiol-oligonucleotides or corresponding MFL2 conjugates were tested on a15% denaturing 8M urea gel, and stained with SYBR-Gold.

FIG. 31 shows LD-TAPER demonstration with Gaussia luciferasesplit-protein fusions with FKBP-F36V-C22S, mediated viaMFL2-oligonucleotide conjugates in Architecture 1; luminescence readingsare shown in the Y-axis; the preparation of mutually complementaryLD-TAPER haplomers giving time-dependent positive signals is boxed inthe Legend.

FIG. 32 shows processes used for LD-TAPER demonstration withArchitecture 2.

FIG. 33 (Panels 1 and 2) shows LD-TAPER demonstration with Gaussialuciferase split-protein fusions with FKBP-F36V, or FKBP-F36V-C22S,mediated via MFL2-oligonucleotide conjugates in Architecture 2 viasolid-phase capture; Panel 1, FKBP-F36V Gaussia fusions, where(A)=N-terminal Gaussia fragment-FKBP-F36V fusion, and(B)=FKBP-F36V-C-terminal Gaussia fusion; 407MFL2 and409MFL2=oligonucleotides 407 and 409 respectively conjugated to compoundMFL2; 407 u and 409 u=corresponding unconjugated thiol-oligonucleotides;Panel 2, FKBP-F36V-C22S Gaussia fusions, where (A)=N-terminal Gaussiafragment-FKBP-F36V-C228 fusion, and (B)=FKBP-F36V-C22S-C-terminalGaussia fusion; 407MFL2, 409MFL2, 407 u, and 409 u as for Panel 1; afterhybridization with desthiobiotinylated template (see, FIG. 32 ),template and template-bound haplomers were isolated on streptavidinmagnetic beads, eluted with free D-biotin, and evaluated for Gaussialuciferase activity after a 30 minute incubation (luminescence units onY-axes).

FIG. 34 shows LD-TAPER demonstration with Gaussia luciferasesplit-protein fusions with FKBP-F36V-C22S, mediated viaMFL2-oligonucleotide conjugates in Architecture 2, with the template orcontrol in solution phase.

DESCRIPTION OF EMBODIMENTS

Certain exemplary embodiments will now be described to provide anoverall understanding of the principles of the structure, function,manufacture, and use of the compositions and methods disclosed herein.One or more examples of these embodiments are illustrated in theaccompanying drawings. Those skilled in the art will understand that thecompositions and methods specifically described herein and illustratedin the accompanying drawings are non-limiting exemplary embodiments andthat the scope of the present disclosure is defined solely by dieclaims. The features illustrated or described in connection with oneexemplary embodiment may be combined with the features of otherembodiments. Such modifications and variations are intended to beincluded within the scope of the present disclosure,

As used herein, the singular forms “a,” and “the” include pluralreferences unless the content clearly dictates otherwise. The terms usedin this disclosure adhere to standard definitions generally accepted bythose having ordinary skill in the art. In case any further explanationmight be needed, some terms have been further elucidated below.

As used herein, the phrase “anti-target loop portion” refers to aportion of a haplomer that facilitates sequence-specific binding to atarget nucleic acid molecule.

As used herein, the term “base” refers to a molecule containing a purineor pyrimidine group, or an artificial analogue, that forms a bindingpair with another corresponding base via Watson-Crick or Hoogsteenbonding interactions. Bases further contain groups that facilitatecovalently joining multiple bases together in a polymer, such as anoligomer. Non-limiting examples include nucleotides, nucleosides,peptide nucleic acid residues, or morpholino residues.

As used herein, the terms “bind,” “binds,” “binding,” and “bound” referto a stable interaction between two molecules that are close to oneanother. The terms include physical interactions, such as chemical bonds(either directly linked or through intermediate structures), as well asnon-physical interactions and attractive forces, such as electrostaticattraction, hydrogen bonding, and van der Waals/dispersion forces.

As used herein, the phrase “bioconjugation chemistry” refers to thechemical synthesis strategies and reagents that ligate common functionalgroups together under mild conditions, facilitating the modularconstruction of multi-moiety compounds.

As used herein, the phrase “chemical linker” refers to a molecule thatbinds one haplomer to another haplomer or one moiety to another moietyon different compounds. A linker may be comprised of branched orunbranched covalently bonded molecular chains

As used herein, the phrase “dosage unit form” refers to physicallydiscrete units suited as u dosages for the subjects to be treated.

As used herein, the term “haplomer” refers to nucleic acid moleculeslinked to a ligand that bind to a target nucleic acid molecule templatein a sequence-specific manner and participate in protein formationduring nucleic acid templated assembly. Also included herein are“derivatives” or “analogs” such as salts, hydrates, solvates thereof, orother molecules that have been subjected to chemical modification andmaintain the same biological activity or lack of biological activity,and/or ability to act as a haplomer, or function in a manner consistentwith a haplomer.

As used herein, the phrase “non-traceless bio-orthogonal chemistry”refers to a reaction involving selectively-reactive moieties in whichpart or all of the structure of the selectively-reactive moieties isretained in the product structure.

As used herein, the phrase “nucleic acid templated assembly” refers tothe dimerization or folding of a protein on a target nucleic acidmolecule, such that the protein formation can be facilitated byhaplomers being assembled in proximity when bound to a target nucleicacid molecule.

As used herein, the terms “oligomer” and “oligo” refer to a moleculecomprised of multiple units where some or all of the units are basescapable of forming Watson-Crick or Hoogsteen base-pairing interactions,allowing sequence-specific binding to nucleic acid molecules in a duplexor multiplex structure. Non-limiting examples include, but are notlimited to, oligonucleotides, peptide nucleic acid oligomers, andmorpholino oligomers.

As used herein, the phrase “pathogenic cell” can refer to a cell that iscapable of causing or promoting a diseased or an abnormal condition,such as a cell infected with a virus, a tumor cell, and a cell infectedwith a microbe, or a cell that produces a molecule that induces ormediates diseases that include, but are not limited to allergy,anaphylaxis, inflammation and autoimmunity.

As used herein, the phrase “pharmaceutically acceptable” refers to amaterial that is not biologically or otherwise unacceptable, that can beincorporated into a composition and administered to a patient withoutcausing unacceptable biological effects or interacting in anunacceptable manner with other components of the composition.

As used herein, the phrase “pharmaceutically acceptable salt” means asalt prepared from a base or an acid which is acceptable foradministration to a patient, such as a mammal (e.g., salts havingacceptable mammalian safety for a given dosage regime).

As used herein, the term “salt” can include salts derived frompharmaceutically acceptable inorganic acids and bases and salts derivedfrom pharmaceutically acceptable organic acids and bases and theirderivatives and variants thereof.

As used herein, the term “sample” refers to any system that haplomerscan be administered into, where nucleic acid templated assembly mayoccur. Examples of samples include, but are not limited to, fixed orpreserved cells, whole organisms, tissues, tumors, lysates, or in vitroassay systems.

As used herein, the phrases “set of corresponding reactants” or“corresponding haplomers” refer to haplomers that come together on asingle target nucleic acid molecule to take part in a templated assemblyreaction.

As used herein, the term “superantigen” refers to an antigen that bindsto a broad subset of T cells that express a particular variable (V)region.

As used herein, the phrase “target compartment” refers to a cell, virus,tissue, tumor, lysate, other biological structure, spatial region, orsample that contains target nucleic acid molecule(s), or a differentamount of target nucleic acid molecules than a non-target compartment.

As used herein, the phrases “target nucleic acid sequence” and “targetnucleic acid molecule” are used interchangeably and refer to a sequenceof units or nucleic acids which are intended to act as a template fornucleic acid templated assembly.

As used herein, the phrase “templated assembly product,” refers to theprotein formed by dimerization or folding of two fragments of aparticular protein associated with the haplomers.

As used herein, the phrase “traceless bio-orthogonal chemistry” refersto a reaction involving haplomer ligands in which a naturally occurringbond, such as an amide, is formed by elimination of part or all of thebio-orthogonal moiety from the ligand structure thus produced.

Nucleic acid molecules that are specific to designated cells of interest(whether these are represented by pathological tumor cells, abnormalimmune cells, or any other cellular types) can be used as templates forthe generation of novel structures (e.g., effector structures) by meansof proximity-induced enhancement of molecular interactions (see, forexample, PCT Publication No. WO 2014/197547). Such templated productscan be designed to trigger cell death in various ways, or to modulatecellular activities. Cell-type specific nucleic acids can be sourcedfrom specific transcribed mRNAs, or via nucleic acid aptamers which canserve to adapt non-nucleic acid targets for the provision of a definedtemplate sequence.

In the original process of templated assembly for diagnostic ortherapeutic purposes described above, reactive groups are brought intospatial proximity by virtue of their linkage with oligonucleotides ofpredetermined sequence, which themselves co-hybridize in proximity on atarget nucleic acid molecule template. The template-directed modifiedoligonucleotides bearing mutually reactive groups are termed“haplomers.” Such enforced proximity of reactive groups greatly enhancesproduct formation, and thus cell-type specific transcripts can directthe production of desired molecules in cells of interest. The generalprinciple of TAPER can be altered to a two-level process, as describedherein, by appending specific ligands to each haplomeric oligonucleotideinstead of directly interactive functional groups. Thus, in the originalconfiguration of TAPER (herein termed “conventional TAPER”), the processcan be signified as occurring within a single reaction sequence, wherethe template can be considered functionally as a specific catalyst:

where H1 and H2 represent haplomers bearing reactive groups A and B,respectively. Upon hybridization to specific template, aproximity-driven reaction intermediate between A and B is formed [A:B],leading rapidly to the formation of product [P].

In a ligand-directed alternate of TAPER (i.e., LD-TAPER), the desiredprocess occurs at two distinct levels:

where H1 and H2 represent haplomers, L1 represents any form of ligand,and D indicates a protein binding domain or any other molecule capableof binding to the ligand in a specific manner. Here, the initial bindingof two molecules of D to the two ligand molecules (displayed in spatialproximity via the haplomer template binding) is shown as a transitionalstate before formation of a D-D dimer, which may or may not bethermodynamically reversible. In the second stage of LD-TAPER, thetemplate is still required to enforce the ligand L1-L1 spatialproximity. This is the case for all variant embodiments of LD-TAPERexcept where a proximity-enhanced covalent interaction is designed tooccur between two modified L1 molecules, or modified L1 and L2molecules, such that they become covalently linked and, thus, stabilizedas a pair. Provided of course that their interactions with cognatebinding domains is not affected by the ligand-ligand covalent joining(as a necessary pre-condition for this embodiment), then the subsequentstage two of the LD-TAPER process becomes template-independent. Thegeneralizable nature of the two-stage LD-TAPER process is schematicallydepicted in FIG. 1 . Referring to FIG. 1 , haplomers with specificappended ligands (L1 and L2) hind to target nucleic acid moleculetemplate such that the ligands fall into spatial proximity with eachother. Proteins or polypeptides (P1 and P2) fused with binding domainsfor the ligands of interest (R1 and R2) are directed to the templatedhaplomer site by the interaction between the ligand and thecorresponding ligand binding domain. The P1 and P2 segments brought intoenforced spatial proximity, promoting dimerization or protein folding.

Although LD-TAPER is a two-stage process, it is not essential inprinciple that the haplomer/template hybridization comprises the firststep (as portrayed in FIG. 1 ). In some embodiments, it may be desirableto initially pre-form each haplomer (bearing an appended ligand) withits designated cognate ligand binding domain, to form haplomer-fusionprotein complexes. Subsequently, the hybridization step is performed,resulting in the desired molecular proximity of protein fragments fusedwith the ligand binding domains themselves. Using the above terminology,this may be represented as:

where the substituents are as described previously.

Various embodiments of LD-TAPER are possible, where H1 and H2 areappended with different ligands (L1 and L2), or where ligands can bebound by two separate binding domains (D1 and D2). If D1 and D2 aresplit-protein polypeptides, the second-level event can be comprised ofmature protein folding between D1 and D2.

Evolution has provided important and very useful examples of naturalsmall-molecule ligands, which can be exploited for biotechnologicalaims. In this respect, some natural small molecules can be noted thathave had a major impact on both experimental and applied immunology. Thenatural product immunosuppressants Cyclosporin A and FK506 (from fungaland bacterial sources, respectively) have revolutionized organtransplantation through their blocking of T cell activation. Thesecompounds bind different cellular proteins, cyclophilin andFK506-binding protein (FKBP) respectively, but they share a commontarget in the form of the protein phosphatase calcineurin, acalcium-responsive regulator of multiple signaling pathways. Thecyclophilin-cyclosporin A and FK506-FKBP complexes, in turn, formternary complexes with calcineurin and inhibit its role in T cellactivation via transcription factors of the NFAT family. Rapamycin,another natural immunomodulatory molecule, binds the same FKBP as theFK506 molecule, but forms a ternary complex with the unrelated proteinFRAP/mTOR with distinct signal pathway roles. The interactions between:a) FK506, calcineurin, and FKBP, and b) rapamycin, FKBP, and mTOR-FRBare depicted in FIG. 2 .

The FK506/FKBP interaction has been exploited in many areas, furtherhelped by the small size of the FKBP binding domain (about 100 aminoacid residues). In order to enhance the potential therapeutic utility ofthis interaction and minimize binding to endogenous FKBP protein, mutantderivatives of FKBP have been derived which preferentially bind alteredFK506 analogs. Thus, the F36V FKBP mutant binds a specific FK506derivative much more strongly than the wild-type molecule itself.

Although small molecule ligands are one type of ligand for use inLD-TAPER, they are not the only type of ligands that can be used. Thereexist relatively small mutually interactive protein domains (e.g.,fragments of proteins) that are applicable in this context, an exampleof which are leucine zippers. Suitable examples of interactive proteindomains are the c-jun and c-fos zipper domains, which generally arepolypeptides of less than 50 amino acid residues, includinghelix-initiating and helix-terminating segments. While c-jun can formhomodimers, c-fos cannot; and c-fos:c-jun heterodimers are significantlymore stable than c-jun:c-jun homodimers. Appending such zipper sequencesto oligonucleotides for the purposes of creating LD-TAPER haplomersprovides each haplomer, with the zipper acting as a ligand, to bind tofusion proteins of desired polypeptides with the complementary zipper asa ligand binding domain.

Whether the ligands used for LD-TAPER are small chemical entities orinteractive protein domains, or any other structure for which acomplementary binding element exists, the two-stage LD-TAPER process canbe applied towards enforced dimerization of either the same partnerprotein fragments (homodimerization) or different partner proteinfragments (heterodimerization). These multiple aspects of LD-TAPER aresummarized in FIG. 3 .

The LD-TAPER processes and components thereof can be generally describedby die following general representations. The embodiments of LD-TAPERusing small molecule ligands may exploit, in a non-limiting example, theFKBP binding domain for FK506. It is known that bivalent analogs ofFK506 can drive the dimerization of FKBP binding domains, or mutantderivatives of them. In turn, if such FKBP domains are fused with otherproteins, the latter themselves undergo a forced dimerization event(see, FIG. 4 ), which in some cases may activate the proteins ofinterest towards potent biological activities.

For the purposes of small molecule LD-TAPER, a monovalent domain bindingcompound is used, which is chemically appended to the 5′ or 3′ ends ofshort oligonucleotide strands, which comprise a portion of the resultinghaplomers. When such haplomers are hybridized to a complementary targetnucleic acid molecule template, the appended monovalent compounds arebrought into spatial proximity (see, FIG. 5 ). Protein ligand bindingdomains which recognize and bind the monovalent compound (i.e., ligand)are likewise brought together close in spatial positioning, as are anyother protein domains fused to the binding domains themselves (see, FIG.5 ). Such enforced dimerization of the fusion domains of the proteins ofinterest leads to functional activation with measurable biologicalconsequences.

Where LD-TAPER is mediated via small molecule ligands, the ligandidentity may correspond to other low molecular weight defined compounds,or natural or artificial peptides, peptidomimetics, or any othermolecule with a defined binding partner. Small molecule LD-TAPER can bedesigned with a number of distinct template:haplomer architectures. Insome embodiments comprising the simplest arrangement, the haplomers aremutually complementary to each other, such that the resulting duplexenforces the desired spatial proximity of the ligand-interacting proteinfusions. This configuration is herein referred to as Architecture 1(see, FIG. 9 ). When the haplomers are not complementary to each other,but hybridize to spatially adjacent sites on a target nucleic moleculetemplate, Architecture 2 is achieved (see, FIG. 5 and FIG. 10 ).Non-contiguous hybridization sites can still be LD-TAPER targets whensuitable configurations exist. Thus, when LD-TAPER haplomers hybridizeto the outer boundaries of a stem loop structure, spatial proximity isachieved, producing Architecture 3 (see, FIG. 11 ). Conversely, theinner region of a stein loop can be potentially targeted if haplomersanneal with the appropriate sites relative to each other, thus producingArchitecture 4 (see, FIG. 12 ).

In some embodiments, the variation of TAPER referred to as “lockedTAPER” is readily applicable to LD-TAPER. For locked LD-TAPER, the firstbottle haplomer and second haplomers are conjugated with a predeterminedligand, in an analogous manner to other LD-TAPER architectures in theabove embodiments. By the nature of the locked TAPER process, thehybridization site for the second haplomer-polypeptide conjugate is notaccessible except in the presence of specific target, wherehybridization occurs with the anti-target loop portion of the firstbottle haplomer. Subsequently, the hybridization site for the secondhaplomer-polypeptide conjugate is rendered accessible, and in turnproximity-promoted ligand-mediated dimerization (see, FIG. 13 ).

Within a locked-TAPER system, when two oligonucleotides bearingpolypeptide conjugates are in hybridization-mediated spatial proximity(see, FIG. 13 ), the structure of the assembly pieces corresponds toArchitecture 1 (see, FIG. 9 ), since the two derivatizedoligonucleotides are complementary to each other, rather thancomplementary to a target nucleic acid molecule template as inArchitectures 2-4. Nevertheless, since the anti-target loop portion of alocked-TAPER first bottle haplomer must hybridize to a target nucleicacid molecule template in order to expose the recognition site for thesecond haplomer, the anti-target loop portion binding to the targetnucleic acid molecule itself can occur via different architectures.Thus, although the target hybridization of the locked TAPERoligonucleotide in FIG. 9 corresponds to Architecture 2 (see, FIG. 5 ),target hybridization by means of Architectures 3 and 4 (see, FIGS. 6 and7 , respectively) are equally possible. Locked TAPER accordingly has theunique feature whereby the TAPER assembly is always constant withArchitecture 1, but target hybridization can assume variablearchitectures. In other words, for conventional TAPER, the targethybridization and assembly-directing hybridizations coincide, but forlocked TAPER they are distinct and separable.

In some embodiments, the ligands for LD-TAPER are not small molecules ina conventional sense, but rather small interactive protein domains.These may include, but are not limited to, interacting leucine zippermotifs, which themselves may be comprised of, but not limited to,parallel zippers such as c-jun:c-fos; mad:max; and c-myc:max, orantiparallel zippers, such as that from Thermus thermophilus seryl-tRNAsynthetase.

In some embodiments, when small interactive protein domains are used asligands, the well-characterized c-jun:c-fos zipper pair is used, asdepicted in FIG. 14 . Haplomers comprised of oligonucleotide segmentscomplementary to a target nucleic acid molecule template of interest maybe conjugated with c-jun domains, and then hybridized with template.Subsequently, protein fragments of interest fused with c-fos domains areadded, leading to complex formation and enforced dimerization of theprotein fragment of interest. In the depiction of FIG. 14 , whichcorresponds to the two-stage strategy generalized with Equation 2.1 and2.2 above, the initial duplex may be further stabilized by the formationof c-jun homodimers. However, since c-jun:c-fos heterodimers aresignificantly more stable, the introduction of the fos-fusion proteinresults in the preferential formation of the desired heterodimericcomplex (c-fos itself cannot form homodimers). In an alternate versionof this embodiment, the haplomers bearing c-jun conjugated tags arepre-assembled with the fos-fusion protein of interest, before adding tothe target system containing the template of interest. This alternatearrangement corresponds to the two-stage strategy generalized withEquation 3.1 and 3.2 above.

In embodiments using small interactive protein domains as ligands, thepolarity of the conjugation of the small domain tag should be taken intoaccount. This can be exemplified with the particular embodiments usingfos-jun heterodimerization, where the leucine zipper interaction occurswith a parallel orientation. If haplomers have appended c-Jun tags suchthat their c-Jun helices are in a parallel orientation followinghybridization (see, FIG. 15 ), then subsequent complex formation withc-fos fusion proteins will orient the fusion in a parallel sense; thereverse situation may disfavor dimerization between the protein segmentsof interest (see, FIG. 15 ). However, for certain other applications ofLD-TAPER (most notably, for the assembly of split-protein fragments, asbelow), an antiparallel orientation may be beneficial. For this reason,it is advantageous if strategies exist for conjugating 5′ or 3′oligonucleotide ends with small protein tags by either their N- orC-termini.

In embodiments using small interactive protein domains as ligands, c-junand c-fos have significant advantages. In both cases, theiralpha-helical zippers are fully defined by relatively shortpolypeptides, neither of which possess internal cysteine residues. Thesesequences can be readily produced by expression systems within E. coil,and are short enough that complete synthesis is feasible. This is usefulfor the c-Jun segment, since it renders thiol-mediated conjugation witholigonucleotides a facile approach. For c-jun tags, the sequence usedherein for making N-terminal conjugates is a 47-mer, where theN-terminal cysteine is shown, and the bold sequences denote helicalboundaries: CSGGASLERIARLEEKVKTIKAQNSELASTANMIRE QVAQLKQKGAP (SEQ IDNO:1). For c-jun tags, the sequence used herein for making C-terminalconjugates is a 49-mer, where the C-terminal cysteine is shown, and thebold sequences denote helical boundaries:SGASLER.IARLEEKVKTLKAQNSEIASTANMLREQVAQLKQK GAPSGGC (SEQ D NO:2). Thesequence of the fos zipper to be made as fusions with the proteinfragment of interest is a 41-mer, where the bold sequences denotehelical boundaries: ASRELTDTLQAETDQLEDEKSALQTEIANLLKEKEKLEGAP (SEQ IDNO:3). Additional extended serine-glycine linkers can be insertedbetween the c-Fos sequence and the protein fragment of interest.

In some embodiments, mutants of c-Jun are used that cannot formhomodimers, but which can still heterodimerize with c-Fos. Such modifiedsequences with N-terminal cysteine residues include, but are not limitedto: CSGGASLERIARLEEKVKSFKAQNSENASTAN MLREQVAQLKQKGAP (SEQ ID NO:4),where bold residues denote changes from wild-type, and double-underlinedsequences denote helical boundaries. Such modified sequences withC-terminal cysteine residues include, but are not limited to:SGASLERIARLEEKVKSFKAQN SENASTANMLREQVAQLKQKGAPSGGC (SEQ ID NO:5), wherebold residues denote changes from wild-type, and double-underlinedsequences denote helical boundaries.

In some embodiments of LD-TAPER using either small molecule ligands orsmall interactive domain ligands, the application may be aimed towardsthe assembly of split-protein fragments. In other LD-TAPER embodiments,the protein fragments fused with ligand binding domains are self-foldinginto well-ordered and stable structures, but this is not the case withLD-TAPER applied towards split-protein assembly. In the latter, theprotein sequences appended to the ligand-binding domains only attaintheir mature folds when they are placed in close spatial proximity inthe correct orientation.

In some embodiments of LD-TAPER for split protein refolding that utilizesmall molecule ligands, the split protein polypeptides are separatelyexpressed as fusions with the FKBP FK506-binding domain. The N-terminalsplit protein fragment is expressed with a C-terminal FKBP segment,while the C-terminal split protein fragment is expressed with anN-terminal FKBP segment (see, FIG. 16 ). Upon binding of the FKBPdomains to templated haplomeric conjugates bearing a monovalentFKBP-binder (see, FIGS. 6-8 ), proximity-enabled folding of the maturepolypeptide is elicited (see, FIG. 16 , depicted for templateArchitecture 2). Because the FKBP domains binding each haplomeric ligandare in the same (parallel) orientation, the split protein fragments areplaced on opposite sides of the spatially proximal FKBP pair (see, FIG.16 ). However, split protein refolding can still occur if the FKBPdomains and the split protein polypeptides are separated by sufficientlylong serine-glycine linkers.

Similar principles apply for embodiments of LD-TAPER for split proteinrefolding that utilize small interactive protein domains as ligands. Insome embodiments using c-Jun:c-Fos interactions, the N-terminal splitprotein fragment is expressed as a C-terminal fusion with c-Fos, whilethe C-terminal split protein fragment is expressed as an N-terminal withc-Fos (see, FIG. 17 ). In these embodiments, an antiparallel arrangementof haplomers tagged with c-Jun can be readily accomplished, which isadvantageous for placement of the protein fragments in juxtaposition onthe same side of the c-Jun pair. Nevertheless, as for the split proteinLD-TAPER mediated by small-molecule ligands (see, FIG. 16 ), parallelc-Jun tags can still be used if a serine-glycine linker of sufficientlength is employed (see, FIG. 17 ). Although the depictions ofsplit-protein embodiments of LD-TAPER use haplomer-template Architecture2 (see, FIG. 10 ), Architectures 3 and 4 (see, FIGS. 11 and 12 ) areequally applicable. Architecture 1 (see, FIG. 9 ) in this contextcorresponds to locked TAPER (see, FIG. 13 also very compatible withsplit-protein embodiments of LD-TAPER.

In the above embodiments (as depicted in FIG. 5 , and FIGS. 9-17 ) bothhaplomers bear a common ligand tag, and proteins or polypeptidefragments of interest are tagged with a common ligand binding domainThis arrangement is well-suited to systems featuring homodimerization,but is not ideal for heterodimerization, by its nature as a two-stageprocess. If a protein heterodimer A-B is to be assembled on a templatewith haplomers H-A and H-B where the first stage is haplomer-templatebinding (as in Equation 2.1 and 2.2 above), and a monoligand/bindingdomain system is used, then the protein segments A and B can assort inthree possible ways, only one of which is the correct A-B:

Equations 4.1, 4.2 (Only the final associative states of A and B areshown for simplicity).

Since the split-protein application of LD-TAPER involves two separatebinding domain-polypeptide conjugates (see, FIGS. 16 and 17 ), theheterodimerization characteristics also apply in this case. While theuse of a monoligand/binding domain system for hetero-components (as forA and B above) is not precluded, it may result in a very significantloss of activity if the two-stage procedure of Equations 4.1 and 4.2 areused. Nevertheless, this characteristic can be overcome in someembodiments by preparing the haplomer/ligand binding domain fusioncomplexes in advance of exposure to the nucleic acid molecule template.Pre-assembly of the haplomer-fusion protein complexes allows controlover which ligand binding domain-fusion proteins form partners with aspecific haplomer. By such means, a defined haplomer sequence carries aspecific ligand binding domain complex of interest, in an analogousmanner to Equations 3.1 and 3.2:

Equations 5.1, 5.2 (Only the final associative states of A and B areshown for simplicity).

In some embodiments of LD-TAPER, it may nonetheless be advantageous tobe able to use a heterocomponent system with full efficiency, withoutthe need for pre-assembly of haplomers and protein fusion domains. Thiscan be achieved by using two distinct ligands, each with a distinct andspecific partner ligand binding domain Such an arrangement can berepresented with the same sythbology as above (where L_(A) and L_(B)represent two distinct ligands with different binding specificities, andA and B represent the corresponding ligand binding entities):

Equations 6.1, 6.2 (Only the final associative states of A and B areshown for simplicity)

In some embodiments of heterocomponent LD-TAPER, the specificity ofleucine zipper interactions is used. These include, but are not limitedto, c-Jun:c-Fos, and c-Myc:Max heterodimer formation (see, FIG. 19 ).Thus, haplomers can be prepared with terminal conjugations with c-Junand c-Myc zippers, and protein domains of interest (or split-proteinpolypeptide fragments) can be expressed as fusions with c-Fos and Max.The templated jun/myc haplomers direct the forced proximity of the twodomains of interest (see, FIG. 19 ), for ensuing dimerization(pre-folded momomeric domains) or folding (split-protein polypeptidefragments).

In some embodiments of LD-TAPER, it may be beneficial to stabilize smallmolecule monovalent ligands as a linked pair after the desiredhaplomeric templating has taken place. Although in many cases, enforceddimerization of polypeptide folding by small-molecule LD-TAPER is eitherirreversible or slowly reversed, the template-mediated conversion of themonovalent ligands to bivalency enables the subsequent dimerization tobecome template-independent, just as it is for conventional bivalentchemical dimerizers in isolation. Cross-linking of haplomeric smallmolecule ligands is effected by equipping the monovalent ligandcompounds with side chains corresponding to bio-orthogonal click groups,as schematically depicted in FIG. 20 . Following the in situ templatedclick reaction, the initially monovalent ligands are converted in effectinto a stable bivalent species, for subsequent interaction withappropriate ligand binding domains (see, FIG. 20 ).

Some embodiments of LD-TAPER involve homo- or hetero-dimerization ofpre-folded proteins that perform important biological functions. Suchproteins may be natural, or artificial constructs. These include, butare not limited to, the iCasp9 fusion protein (an artificial constructwith a modified Caspase-9 sequence fused with a mutant FKBP sequence,proapoptotic proteins, or dimeric transcription factors. The effects ofenforced dimerization mediated by LD-TAPER may be gauged, in variousembodiments, by apoptotic assays, activation of reporter genes, orgeneration of specific fluorescence.

The LD-TAPER processes and components thereof can be generally describedby the following more specific embodiments.

The present disclosure provides haplomer-ligand complexes comprising: a)a haplomer, wherein the haplomer comprises a polynucleotide that issubstantially complementary to a target nucleic acid molecule; and b) aligand linked to the 5′ or 3′ terminus of the haplomer, wherein theligand comprises a ligand partner binding site. In some embodiments, thepolynucleotide of the haplomer comprises from about 6 to about 20nucleotide bases. In some embodiments, the the polynucleotide of thehaplomer comprises from about 8 to about 15 nucleotide bases.

In some embodiments, a pair of haplomer-ligand complexes works intandem. In some embodiments, the ligand of the first haplomer-ligandcomplex is linked to the 5′ terminus of the polynucleotide of the firsthaplomer-ligand complex, and the ligand of the second haplomer-ligandcomplex is linked to the 3′ terminus of the polynucleotide of the secondhaplomer-ligand complex.

In some embodiments, the polynucleotide of the first haplomer-ligandcomplex is substantially complementary to the polynucleotide of thesecond haplomer-ligand complex. In some embodiments, the polynucleotideof the first haplomer-ligand complex is substantially complementary to atarget nucleic acid molecule, and the polynucleotide of the secondhaplomer-ligand complex is substantially complementary to the targetnucleic acid molecule at a site in spatial proximity to thepolynucleotide of the first haplomer-ligand complex.

In any of the embodiments described herein, the haplomer-ligandcomplexes are in spatial proximity (when bound to a target nucleic acidmolecule) such that the ligands, and hence their respective ligandbinding domains, can properly interact to induce the folding ordimerization of their respective fragments of the protein of interest.Thus, for any haplomer-ligand pairs, reactivity can occur where the gapN between the first and second haplomer-ligand complex binding to thetarget nucleic acid molecule is 0 (i.e., the haplomer-ligand complexesare immediately juxtaposed), and progressively greater gaps (N>0) willprogressively diminish activity. Thus, in some embodiments, there is 0nucleotides between the binding of a first haplomer-ligand complex andsecond haplomer-ligand complex to the target nucleic acid molecule. Insome embodiments, there is less than 6 nucleotides between the bindingof a first haplomer-ligand complex and second haplomer-ligand complex tothe target nucleic acid molecule. In some embodiments, there is lessthan 5 nucleotides between the binding of a first haplomer-ligandcomplex and second haplomer-ligand complex to the target nucleic acidmolecule. In some embodiments, there is less than 4 nucleotides betweenthe binding of a first haplomer-ligand complex and secondhaplomer-ligand complex to the target nucleic acid molecule. In someembodiments, there is less than 3 nucleotides between the binding of afirst haplomer-ligand complex and second haplomer-ligand complex to thetarget nucleic acid molecule. In some embodiments, there is less than 2nucleotides between the binding of a first haplomer-ligand complex andsecond haplomer-ligand complex to the target nucleic acid molecule.

In some embodiments, both ligands are small molecule ligands or bothligands are interactive protein domains. In some embodiments, the ligandof the first haplomer-ligand complex further comprises a bio-orthogonalmoiety, and the ligand of the second haplomer-ligand complex furthercomprises a bio-orthogonal moiety, wherein the bio-orthogonal moiety ofthe first haplomer-ligand complex is reactable with the bio-orthogonalmoiety of the second haplomer-ligand complex.

The present disclosure also provides bottle haplomer-ligand complexescomprising: a) a bottle haplomer, wherein the bottle haplomer comprisesa polynucleotide, wherein the polynucleotide comprises: i) a first stemportion comprising from about 10 to about 20 nucleotide bases; ii) ananti-target loop portion comprising from about 16 to about 40 nucleotidebases and having a first end to which the first stem portion is linked,wherein the anti-target loop portion is substantially complementary to atarget nucleic acid molecule; and iii) a second stem portion comprisingfrom about 10 to about 20 nucleotide bases linked to a second end of theanti-target loop portion, wherein the first stem portion issubstantially complementary to the second stem portion; and b) a ligandlinked to the terminal end of either the first stem portion or thesecond stem portion, wherein the ligand comprises a ligand partnerbinding site; wherein the T_(m) of the anti-target loop portion:targetnucleic acid molecule is greater than the T_(m) of the first stemportion:second stem portion.

In some embodiments, the first stem portion that comprises from about 10to about 20 nucleotide bases. In some embodiments, the first stemportion comprises from about 1:2 to about 18 nucleotide bases.

In some embodiments, the anti-target loop portion comprises from about16 to about 40 nucleotide bases. In some embodiments, the anti-targetloop portion comprises from about 18 to about 35 nucleotide bases. Theanti-target loop portion has a first end to which the first stem portionis linked. The anti-target loop portion is substantially complementaryto a target nucleic acid molecule. In some embodiments, a ligand islinked to the second stem portion.

In some embodiments, the anti-target loop portion can further comprisean internal hinge region, wherein the hinge region comprises one or morenucleotides that are not complementary to the target nucleic acidmolecule. In some embodiments, the hinge region comprises from about 1nucleotide to about 6 nucleotides, from about 1 nucleotide to about 5nucleotides, from about 1 nucleotide to about 4 nucleotides, from about1 nucleotide to about 3 nucleotides, or 1 or 2 nucleotides.

In some embodiments, the second stem portion comprises from about 10 toabout 20 nucleotide bases. In some embodiments, the second stem portioncomprises from about 12 to about 18 nucleotide bases. The second stemportion is linked to a second end of the anti-target loop portion. Thefirst stem portion is substantially complementary to the second stemportion. In some embodiments, a ligand is linked to the second stemportion.

In some embodiments, the bottle haplomer comprises the nucleotidesequence 5′-ACTCGAGACGTCTCCTTGTCTTTGCTTTTCTTCAGGACACAGTGGCGAGACOTCTCGAGT-3′ (SEQ IDNO:6) or 5′-ACTCGAGACGTCTCCTTCCTGCCCCTCCTCCTGCTCCGAGACGTC TCGAGT-3′ (SEQID NO:7).

For the polynucleotides of the bottle haplomers described herein, thelength of the particular polynucleotide or portion thereof is lessimportant than the T_(m) of the duplex formed by the interaction of thepolynucleotide, or portion thereof, with another nucleic acid molecule,or portion thereof. For example, the T_(m) of the duplex formed by theinteraction of the anti-target loop portion with the target nucleic acidmolecule (e.g., anti-target loop portion:target nucleic acid molecule)is greater than the T_(m) of the duplex formed by the interaction of thefirst stem portion with the second stem portion (e.g., first stemportion:second stem portion). In some embodiments, the T_(m) of thefirst stem portion:second stem portion subtracted from the T_(m) of theanti-target loop portion:target nucleic acid molecule is from about 10°C. to about 40° C. In some embodiments, the T_(m) of the first stemportion:second stem portion subtracted from the T_(m) of the anti-targetloop portion:target nucleic acid molecule is from about 10° C. to about20° C. In some embodiments, the T_(m) of the first stem portion:secondstem portion is from about 40° C. to about 50° C. In some embodiments,the T_(m) of the anti-target loop portion:target nucleic acid moleculeis from about 60° C. to about 80° C.

In addition, translating the T_(m) information above into specificlengths of the nucleic acid molecules described herein can also dependon the GC content of each nucleic acid molecule. For example, the lengthof a suitable HPV model target nucleic acid molecule is 30 bases (havinga T_(m) of 70° C.), while that for the EBV model target nucleic acidmolecule is only 21 bases (haying a T_(m) of 69° C.), owing to itsgreater %GC.

In some embodiments, a bottle haplomer-ligand complex works in tandemwith a second haplomer-ligand complex. In some embodiments, the bottlehaplomer-ligand complex is any bottle haplomer-ligand complex describedherein, and the second haplomer-ligand complex is any of thehaplomer-ligand complexes described herein. In some embodiments, thesecond haplomer-ligand complex comprises: a) a nucleotide portioncomprising from about 6 to about 20 nucleotide bases that issubstantially complementary to the stem portion of the bottlehaplomer-ligand complex that is linked to the ligand of the bottlehaplomer-ligand complex; and b) a ligand linked to the 5′ or 3′ terminusof the nucleotide portion of the second haplomer-ligand complex, whereinthe ligand comprises a ligand partner binding site; wherein the T_(m) ofthe second haplomer-ligand complex:first or second stem portion linkedto the ligand of the bottle haplomer-ligand complex is less than orequal to the T_(m) of the first stem portion:second stem portion of thebottle haplomer-ligand complex.

In some embodiments, the T_(m) of the duplex formed by the interactionof the second haplomer-ligand complex with either the first stem portionor the second stem portion, whichever stem portion is linked to theligand (e.g., second haplomer-ligand complex:first or second stemportion linked to the ligand), is less than or equal to the T_(m) of thefirst stem portion:second stem portion. In some embodiments, the T_(m)of the duplex formed by the second haplomer-ligand complex and the firstor second stem portion linked to the ligand subtracted from the T_(m) ofthe first stem portion:second stem portion is from about 0° C. to about20° C. In some embodiments, the T_(m) of the duplex formed by the secondhaplomer-ligand complex and the first or second stem portion linked tothe ligand subtracted from the T_(m) of the first stem portion:secondstem portion is from about 5° C. to about 10° C. In some embodiments,the. T_(m) of the duplex formed by the second haplomer-ligand complexand the first or second stem portion linked to the ligand is from about30° C. to about 40° C.

This structural arrangement is designed such that in the absence oftarget nucleic acid molecule template, the locked first haplomer bottledoes not significantly hybridize to its complementary second haplomerand, thus, template-directed product assembly is not promoted under suchconditions. When the specific target nucleic acid molecule template ispresent, on the other hand, the bottle haplomer-ligand complex is“unlocked” by the formation of a more stable hybrid between theanti-target loop region of the bottle haplomer and the target nucleicacid molecule itself. Once this occurs, the first stem portion of thebottle haplomer that is linked to the ligand is free to hybridize to theavailable second haplomer-ligand complex, with resulting proximitybetween the ligands on both.

In any of the haplomer polynucleotides described herein, or any portionthereof, the nucleotide bases are selected from the group consisting ofDNA nucleotides, RNA nucleotides, phosphorothioate-modified nucleotides,2-O-alkylated RNA nucleotides, halogenated nucleotides, locked nucleicacid nucleotides (LNA), peptide nucleic acids (PNA), morpholino nucleicacid analogues (morpholinos), pseudouridine nucleotides, xanthinenucleotides, hypoxanthine nucleotides, 2-deoxyinosine nucleotides, DNAanalogs with L-ribose (L-DNA), Xeno nucleic acid (XNA) analogues, orother nucleic acid analogues capable of base-pair formation, orartificial nucleic acid analogues with altered backbones, or anycombination or mixture thereof.

For any of the any of the haplomer polynucleotides described herein, thecomplementarity with another nucleic acid molecule can be 100%. In someembodiments, one particular nucleic acid molecule can be substantiallycomplementary to another nucleic acid molecule. As used herein, thephrase “substantially complementary” means from 1 to 10 mismatched basepositions, from 1 to 9 mismatched base positions, from 1 to 8 mismatchedbase positions, from 1 to 7 mismatched base positions, from 1 to 6mismatched base positions, from 1 to 5 mismatched base positions, from 1to 4 mismatched base positions, from 1 to 3 mismatched base positions,and 1 or 2 mismatched base positions. In some embodiments, it idesirable to avoid reducing the T_(m) of the anti-target loopportion:target nucleic acid molecule by more than 10% via mismatchedbase positions. The bottle haplomer stem is designed with respect to asecond haplomer, and its structure is deliberately arranged to besomewhat more stable than the formation of the second haplomer duplex.

In some embodiments, the portion of the bottle haplomer-ligand complexthat is not linked to a ligand can have additional nucleotide bases thatoverhang and do not form a part of the stem structure. In someembodiments, the end of the second haplomer-ligand complex that is notlinked to a ligand can have additional nucleotide bases that overhangand do not form a complementary part of the structure with the stemportion of the bottle haplomer-ligand complex. In addition, in someembodiments, the portion of the stem that is linked to the ligand canalso have nucleotide bases that are not base paired with the first stemportion. Such an extension of the stem with a non-hybridized “arm”places the two ligands at a greater spatial distance, thus, tending toreduce their mutual reactivity. So, for a few nucleotide bases (lessthan 10 or less than 5), enforced reactivity is still likely to occur,but will tend to diminish as the non-base paired segment grows inlength.

In some embodiments, added nucleotide bases can be of indefinite length,as long as they did not: 1) have significant homologies with any of theother regions of the locked TAPER oligonucleotides, and thus tend tocross-hybridize and interfere; or 2) interfere non-specifically with anyother features of the system. For example, a long appended sequencemight reduce transformation efficiencies of locked TAPERoligonucleotides used in a therapeutic context. Also, appended sequencesshould be designed to avoid spurious hybridizations with other cellulartranscripts. Appended non-homologous sequences of 20-30 nucleotide basesare suitable. The appended nucleic acid sequences may contain primersequences commonly used in the art. Such examples may include, but arenot limited to, M13, T3, T7, SP6, VF2, VR, modified versions thereof,complementary sequences thereof, and reverse sequences thereof. Inaddition, custom primer sequences are also included. Such primersequences can be used, for example, the possible application ofchemically-ligated oligonucleotides spatially elicited (CLOSE) to thelocked TAPER strategy, (see, PCT Publication WO 2016/89958; which isincorporated herein by 30 reference in its entirety).

In some embodiments, both ligands are small molecule ligands or bothligands are interactive protein domains. In some embodiments, theN-terminus of the interactive protein domain of the bottlehaplomer-ligand complex is linked to the 5′ terminus of thepolynucleotide of the bottle haplomer-ligand complex, and the N-terminusof the interactive protein domain of the second haplomer-ligand complexis linked to the 3′ terminus of the polynucleotide of the secondhaplomer-ligand complex. In some embodiments, the C-terminus of theinteractive protein domain of the bottle haplomer -ligand complex islinked to the 5′ terminus of the polynucleotide of the bottlehaplomer-ligand complex, and the C-terminus of the interactive proteindomain of the second haplomer-ligand complex is linked to the 3′terminus of the polynucleotide of the second haplomer-ligand complex.

In some embodiments, the C-terminus of the interactive protein domain ofthe bottle haplomer-ligand complex is linked to the 5′ terminus of thepolynucleotide of the bottle haplomer-ligand complex, and the N-terminusof the interactive protein domain of the second haplomer-ligand complexis linked to the 3′ terminus of the polynucleotide of the secondhaplomer-ligand complex; or the N-terminus of the interactive proteindomain of the bottle haplomer-ligand complex is linked to the 5′terminus of the polynucleotide of the bottle haplomer-ligand complex,and the C-terminus of the interactive protein domain of the secondhaplomer-ligand complex is linked to the 3′ terminus of thepolynucleotide of the second haplomer-ligand complex.

Any of the bottle haplomers described herein, or any portion thereof,can further comprise a linker between any one or more of the first stemportion and the anti-target loop portion, between the anti-target loopportion and the second stem portion, between the second stem portion andthe ligand, between the first stem portion and the ligand, or betweenthe second haplomer and its ligand. In some embodiments, the linker isselected from the group consisting of an alkyl group, an alkenyl group,an amide, an ester, a thioester, a ketone, an ether, a thioether, adisulfide, an ethylene glycol, a cycloalkyl group, a benzyl group, aheterocyclic group, a maleimidyl group, a hydrazone, a urethane, azoles,an imine, a haloalkyl, and a carbamate, or any combination thereof.

In some embodiments, the second haplomer-ligand complex comprises thenucleotide sequence 5′-AGCTCTCGAGT-3′ (SEQ ID NO:8), or5′-GACGTCTCGAGT-3′ (SEQ ID NO:9).

In any of the embodiments described herein, the ligand is a smallmolecule ligand or an interactive protein domain.

In some embodiments, the ligand is a small molecule ligand. In someembodiments, the small molecule ligand is less than about 2500 Daltons.In some embodiments, the small molecule ligand is a small molecule, apeptide having less than about 20 amino acid residues, a naturally- orartificially-modified peptide, a peptidomimetic, a glycan, an organicenzyme cofactor, or an artificially-derived small molecular ligand. Insome embodiments, the small molecule ligand is derived from compoundsdesigned to target FKBP. In some embodiments, the small molecule ligandis an FKM monovalent ligand.

In some embodiments, the small molecule ligand is derived from compoundsdesigned to target mutant versions of FKBP (Clackson et al., Proc. Natl.Acad. Sci. USA, 1998, 95, 103437-10432). One such modification isFKM-NHS, which comprises a modified monovalent mutant FKBP bindingmoiety with an appended amide, 3-carbon spacer, and a carboxylic acidgroup, esterified with N-hydroxysuccinimide (NHS) (see, FIG. 6 ), whichcan be used, for example, for coupling to amino-labeledoligonucleotides. The spacer moiety of FKM-NHS may comprise, but notlimited to, 4, 5 or 6 carbon atoms in length. FKM-NHS may also bemodified to enhance solubility.

In some embodiments, the small molecule ligand is FKM-sulfo-NHS (see,FIG. 7 ), which can be used, for example, for coupling to amino-labeledoligonucleotides, and with an additional sulfo-group for solubilityenhancement. The NHS moiety carries a sulfo-group. The spacer moiety ofFKM-sulfo-NHS may comprise, but not limited to, 4, 5 or 6 carbon atomsin length.

In some embodiments, the small molecule ligand is comprises anothersolubility-enhancing modification of FKM-NHS, where the spacer arm isconverted into a short segment of polyethylene glycol (PEG) to provideFKM-PEG3-NHS (see, FIG. 8 ), which can be used, for example, forcoupling to amino-labeled oligonucleotide, and with a PEG spacer forsolubility enhancement. The spacer arm of FKM-PEG3-NHS may comprise, butnot limited to, 1, 2, 4, 5, or 6 copies of the monomer ethylene glycol.FKM-NHS and all derivatives of it can be readily and directly coupled toamino-labeled oligonucleotides, at either 5′ or 3′ ends. In addition,the site of appending the reactive group to the PEG chain can be varied.Thus, if the carbon atoms in the PEG chain are numbered, the reactivegroup could be positioned at any of these sites.

In some embodiments, the FKM monovalent ligand is FKM-NHS,FKM-sulfo-NHS, FKM-PEG3-NHS, or monovalent FKBP Ligand-2 (MFL2),wherein: FKM-NHS is

where m is from 3 to 6; FKM-sulfo-NHS is

where m is from 3 to 6; FKM-PEG3-NHS is

where n is from 1 to 6; and MFL2 is

In some embodiments, the ligand is an interactive protein domain. Insome embodiments, the interactive protein domain comprises less than 100amino acid residues. In some embodiments, the interactive protein domainis a leucine zipper domain. In some embodiments, the interactive proteindomain is a c-jun domain, a c-fos domain, a c-myc domain, a c-maxdomain, an NZ domain, or a CZ domain.

In some embodiments, the NZ domain comprises the amino acid sequenceALKKELQ ANKKELAQLKWELQALKKELAQ (SEQ ID NO:10), and the CZ domaincomprises the amino acid sequence EQLEKKLQALEKKLAQLEWKNQALEKKLAQ (SEQ DNO:11).

In some embodiments, the N-terminus of the c-jun domain is linked to the5′ or 3′ terminus of the polynucleotide of the haplomer. In someembodiments, the c-jun domain comprises the amino acid sequenceCSGGASLERIARLEEKVKTILKAQNSELASTANMLR EQVAQLKQKGAP (SEQ ID NO:1),CSGGASLERIARLEEKVKSFKAQNSENASTAN MLREQVAQLKQKGAP (SEQ ID NO:4), orCSGASLERIARLEEKVKSFKAQNSENAS TANMLREQVAQLKQKGAP (SEQ ID NO:12). In someembodiments, the C-terminus of the c-jun domain is linked to the 5′ or3′ terminus of the polynucleotide of the haplomer. In some embodiments,the c-jun domain comprises the amino acid sequence SGASLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKGAPSGGC (SEQ ID NO:2), or SGASLERIARLEEKVKSFKAQNSENASTANMLREQVAQLKQKQAPSGGC (SEQ NO:5).

In some embodiments, the c-Fos domain comprises the amino acid sequence

(SEQ ID NO: 3) ASRELTDTLQAETDQLEDEKSALQTEIANLLKEKEKLEGAP or(SEQ ID NO: 13) SGASRELTDTLQAETDQLEDEKSALQTEIANLLKEKEKLEGAP.

In some embodiments where two haplomer-ligand complexes work in tandem,or where a bottle haplomer-ligand complexes work in tandem with a secondhaplomer-ligand complex, both ligands are either small molecule ligandsor both ligands are interactive protein domains. In some embodiments,both interactive protein domains are leucine zipper domains.

In some embodiments, the N-terminus of the interactive protein domain ofthe first haplomer-ligand complex is linked to the 5 terminus of thepolynucleotide of the first haplomer-ligand complex, and the N-terminusof the interactive protein domain of the second haplomer-ligand complexis linked to the 3′ terminus of the polynucleotide of the secondhaplomer-ligand complex. In some embodiments, the C-terminus of theinteractive protein domain of the first haplomer-ligand complex islinked to the 5′ terminus of the polynucleotide of the firsthaplomer-ligand complex, and the C-terminus of the interactive proteindomain of the second haplomer-ligand complex is linked to the 3′terminus of the polynucleotide of the second haplomer-ligand complex.

In some embodiments, the C-terminus of the interactive protein domain ofthe first haplomer-ligand complex is linked to the 5′ terminus of thepolynucleotide of the first haplomer-ligand complex, and the N-terminusof the interactive protein domain of the second haplomer-ligand complexis linked to the 3′ terminus of the polynucleotide of the secondhaplomer-ligand complex; or the N-terminus of the interactive proteindomain of the first haplomer-ligand complex is linked to the 5′ terminusof the polynucleotide of the first haplomer-ligand complex, and theC-terminus of the interactive protein domain of the secondhaplomer-ligand complex is linked to the 3′ terminus of thepolynucleotide of the second haplomer-ligand complex.

In some embodiments, the C-terminus of the c-jun domain is linked to the5′ or 3′ terminus of the polynucleotides of either or both of the firsthaplomer-ligand complex and second haplomer-ligand complex.

In some embodiments, the ligand linked to the polynucleotide of thefirst haplomer-ligand complex is a c-jun domain or a c-myc domain, andthe ligand linked to the polynucleotide of the second haplomer-ligandcomplex is a c-jun domain or a c-myc domain. In some embodiments, theligand linked to the polynucleotide of the first haplomer-ligand complexor second haplomer-ligand complex is a c-jun domain, and the ligandlinked to the polynucleotide of the other of the first haplomer-ligandcomplex and second haplomer-ligand complex is a c-myc domain. In someembodiments, the ligand linked to the polynucleotide of the firsthaplomer-ligand complex is a c-jun domain, and the ligand linked to thepolynucleotide of the second haplomer-ligand complex is a c-jun domain.

In some embodiments, the ligand linked to the polynucleotide of thebottle haplomer-ligand complex is a c-jun domain, and the ligand linkedto the polynucleotide of the second haplomer-ligand complex is a c-jundomain. In some embodiments, the ligand linked to the polynucleotide ofone of the bottle haplomer-ligand complex and second haplomer-ligandcomplex is a c-jun domain, and the ligand linked to the polynucleotideof the other of the bottle haplomer-ligand complex and secondhaplomer-ligand complex is a c-myc domain.

In some embodiments, the polynucleotide of the bottle haplomer-ligandcomplex comprises the nucleotide sequence of5′-ACTCGAGACGTCTCCTTGTCTTTGCTTTTCTT CAGGACACAGTGGCGAGACGTCTCGAGT-3′ (SEQID NO:6), and the polynucleotide of the second haplomer-ligand complexcomprises the nucleotide sequence of 5′-AGCTCTCGA GT-3′ (SEQ ID NO:8);or the polynucleotide of the bottle haplomer-ligand complex comprisesthe nucleotide sequence of 5′-ACTCGAGACGTCTCCTTCCTGCCCCTCCTCCTGCTCCGAGACGTCTCGAGT-3′ (SEQ ID NO:7), and the polynucleotide of the secondhaplomer-ligand complex comprises the nucleotide sequence5′-GACGTCTCGAGT-3′ (SEQ ID NO:9).

The target nucleic acid molecules that serve as templates in theembodiments described herein can be comprised of any desired nucleicacid sequence capable of hybridizing with the polynucleotides of thehaplomers or the anti-target loop portion of a bottle haplomer. Anysingle-stranded nucleic acid molecule with an accessible sequence ispotentially targetable. These include, but are not limited to, cellularRNAs, mRNA, genomic or organellar DNA, episomal or plasmid DNA, viralDNA or RNA, miRNA, rRNA, snRNA, tRNA, short and long non-coding RNAs,and any artificial sequences used for templating purposes, or any otherbiological or artificial nucleic acid sequence. Artificial sequencesinclude, but are not limited to, aptamers and macromolecular-nucleicacid conjugates. Aptamer templates are also included, where these aredesigned to convert a non-nucleic acid cellular product into atargetable sequence for any form of TAPER, including locked TAPER. Insome embodiments, the target nucleic acid molecule hybridization site iskept as short as possible while: 1) maintaining specificity within acomplex transcriptome or other complex targets; and 2) maintaining thelocked TAPER design guidelines described herein.

Any cell, virus, tissues, spatial regions, lysate, or other subcomponentof a sample that contains a nucleic acid molecule can provide the targetnucleic acid molecule. Target compartments that contain the targetnucleic acid molecule can include, but are not limited to, pathogeniccells, cancer cells, viruses, host cells infected by a virus or otherpathogen, or cells of the immune system that are contributing toautoimmunity such as cells of the adaptive or innate immune systems,transplant rejection, or an allergic response. In some embodiments, atarget nucleic acid molecule can be present in a virus or cell infectedby a virus, but absent in healthy cells. Examples of virus include, butare not limited to, DNA viruses, RNA viruses, or reverse transcribingviruses. In some embodiments, a target nucleic acid molecule can bepresent in a tumor or cancerous cell, but absent in healthy cells.Examples of cancers include, but are not limited to, those caused byoncoviruses, such as the human papilloma viruses, Epstein-Barr virus,hepatitis B virus, hepatitis C virus, human T-lymphotropic viruses,Merkel cell polyoma virus, and Kaposi's sarcoma-associated herpesvirus.In some embodiments, a target nucleic acid molecule can be present in aninfectious agent or microbe, or a cell infected by an infectious agentor microbe but is absent in healthy cells. Examples of infectious agentsor microbes include, but are not limiedt to, viruses, bacteria, fungi,protists, prions, or eukaryotic parasites.

The target nucleic acid molecule can also be a fragment, portion or partof a gene, such as an oncogene, a mutant gene, an oncoviral gene, aviral nucleic acid sequence, a microbial nucleic acid sequence, adifferentially expressed gene, and a nucleic acid gene product thereof.

Examples of cancer-specific target nucleic acids include, but are notlimited to, mutant oncogenes, such as mutated ras, HRAS, KRAS, NRAS,BRAF, EGFR, FLT1, FLT4, KDR, PDGFRA, PDGFRB, ABL1, PDGFB, MYC, CCND1,CDK2, CDK4,or SRC genes; mutant tumor suppressor genes, such as TP53,TP63, TP73, MDM1, MDM2, ATM, RB1, RBL1, RBL2, PTEN, APC, DCC, WT1, IRF1,CDK2AP1, CDKN1A, CDKN1B, CDKN2A, TRIM3, BRCA1, or BRCA2 genes; and genesexpressed in cancer cells, where the gene may not be mutated orgenetically altered, but is not expressed in healthy cells of a sampleat the time of administration, such as carcinoembryonic antigen.

In some embodiments, the target nucleic acid molecule can be present ina differential amounts or concentrations in the target compartments ascompared to the non-target compartments. Examples include, but are notlimited to, genes expressed at a different level in cancer cells than inhealthy cells, such as myc, telomerase, HER2, or cyclin-depedentkinases. In some embodiments, the target nucleic acid molecule can be agene that is at least 1.5X-fold differentially expressed in the targetversus the non-target compartments. Some examples of these include, butare not limited to, genes related to mediating Type I allergicresponses, for which target RNA molecules contain immunoglobulin epsilonheavy chain sequences; genes expressed in T cell subsets, such asspecific T cell receptors (TCRs) which recognize self-antigens in thecontext of particular major histocompatibility (MHC) proteins likeproinsulin-derived peptide and clonally-specific mRNAs containing α or βvariable-region sequences, derived from diabetogenic CD8+ T cells; andcytokines whose production may have adverse outcomes throughexacerbation of inflammatory responses including, but not limited to,TNF-alpha, TNF-beta, IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-15,IL-17, IL-18, IL-21, IL-22, IL-27, IL-31, IFN-gamma, OSM, and LIF.

In some embodiments, a target nucleic acid molecule is present in targetcompartments and an acceptable subgroup of non-target compartments, butnot in a different or distinct subgroup of non-target compartments.Examples include, but are not limited to, genes expressed in cancercells and limited to classes of healthy cells, such as cancer-testisantigens, survivin, prostate-specific antigen, carcinoembryonic antigen(CEA), alpha-fetoprotein and other onco-fetal proteins. Also, manytissues and organs are not essential to otherwise healthy life in theface of serious disease. For example, melanocyte antigens, such asMelan-A/MART-1 and gp100 are expressed on many malignant melanomas aswell as normal melanocytes, and therapies that target these antigens candestroy both tumors and normal melanocytes, resulting in vitiligo, butmajor tumor reduction. Likewise, the reproductive organs may besurgically removed, such as testis, ovary and uterus, as well asassociated organs such as breast and prostate may be targeted whentumors of these tissues arise, and destruction of normal tissues withinthese organs may be a tolerable consequence of therapy. Furthermore,some cells that produce hormones, such as thyroxine and insulin can bereplaced with the relevant protein, allowing potential targeting ofnormal cells that may exist in the presence of tumors of these origins.

Target nucleic acid molecules can also include novel sequences, notpreviously identified. In some embodiments, a sample or samples can beevaluated by sequence analysis, such as next-generation sequencing,whole-transcriptome (RNA-seq) or whole-genome sequencing, microarrayprofiling, serial analysis of gene expression (SAGE), to determine thegenetic makeup of the sample. Target nucleic acid molecules can beidentified as those present in target compartments, but not present innon-target compartments, or present in differential amounts orconcentrations in target compartments as compared to non-targetcompartments. Sequences identified by these methods can then serve astarget nucleic acid molecules.

In some embodiments, when the ligand is a small molecule ligand, thesmall molecule ligand may further comprise a bio-orthogonal moiety. Insome embodiments where two haplomer-ligand complexes work in tandem, theligand of the first haplomer-ligand and complex further comprises abio-orthogonal moiety, and the ligand of the second haplomer-ligandcomplex further comprises bio-orthogonal moiety, wherein thebio-orthogonal moiety of the first haplomer-ligand complex is reactablewith the bio-orthogonal moiety of the second haplomer-ligand complex. Insome embodiments where a bottle haplomer-ligand complex work in tandemwith a second haplomer-ligand complex, the ligand of the bottlehaplomer-ligand complex further comprises a bio-orthogonal moiety, andthe ligand of the second haplomer-ligand complex further comprises abio-orthogonal moiety, wherein the bio-orthogonal moiety of the bottlehaplomer-ligand complex is reactable with the bio-orthogonal moiety ofthe second haplomer-ligand complex.

A bio-orthogonal moiety includes those groups that can undergo “click”reactions between azides and alkynes, traceless or non-tracelessStaudinger reactions between azides and phosphines, and native chemicalligation reactions between thioesters and thiols. Additionally, thebio-orthogonal moiety can be any of an azide, a cyclooctyne, a nitrone,a norbornene, an oxanorbornadiene, a phosphine, a dialkyl phosphine, atrialkyl phosphine, a phosphinothiol, a phosphinophenol, a cyclooctene,a nitrile oxide, a thioester, a tetrazine, an isonitrile, a tetrazole, aquadricyclane, and derivatives thereof. Bio-orthogonal moieties ofmembers of a set of corresponding haplomers are selected such that theywill react with each other.

Multiple bio-orthogonal moieties can be used with the methods andcompositions disclosed herein, some non-limiting examples include:

Azide-Alkene “Click Chemistry”

Click chemistry is highly selective as neither azides nor alkynes reactwith common biomolecules under typical conditions. Azides of the formR—N₃ and terminal alkynes of the form R—C≡CH or internal alkynes of theform R—C≡C—R react readily with each other to produce Huisgencycloaddition products in the form of 1,2,3-triazoles.

Azide-based haplomers have the substructure: R—N₃, where R is a chemicallinker, nucleic acid recognition moiety (e.g. a portion of anoligonucleotide that is complementary to another portion of a nucleicacid molecule), or ligand. Azides and azide derivatives may be readilyprepared from commercially available reagents.

Azides can also be introduced to a ligand during synthesis of theligand. In some embodiments, an azide group is introduced into a peptideligand by incorporation of a commercially available azide-derivatizedstandard amino acid or amino acid analogue during synthesis of theligand using standard peptide synthesis methods. Amino acids may bederivatized with an azide replacing the α-amino group, affording astructure of the form:

where R is a side chain of a standard amino acid or non-standard aminoacid analogue.

Commercially available products can introduce azide functionality asamino acid side chains, resulting in a structure of the form:

where A is any atom and its substituents in a side chain of a standardamino acid or non-standard amino acid analogue.

An azide may also he introduced into a peptide ligand after synthesis byconversion of an amine group on the peptide ligand to an azide bydiazotransfer methods. Bioconjugate chemistry can also be used to joincommercially available derivatized azides to chemical linkers, nucleicacid recognition moieties, or ligands that contain suitable reactivegroups.

Standard alkynes can be incorporated into a haplomer by methods similarto azide incorporation. Alkyne-functionalized nucleotide analogues arecommercially available, allowing alkyne groups to be directlyincorporated at the time of nucleic acid recognition moiety synthesis.Similarly, alkyne-deriviatized amino acid analogues may he incorporatedinto a ligand by standard peptide synthesis methods. Additionally,diverse functionalized alkynes compatible with bioconjugate chemistryapproaches may be used to facilitate the incorporation of alkynes toother moieties through suitable functional or side groups.

Azide-Activated Alkane “Click Chemistry”

Standard azide-alkyne chemistry reactions typically require a catalyst,such as copper(I). Since copper(I) at catalytic concentrations is toxicto many biological systems, standard azide-alkyne chemistry reactionshave limited uses in living cells. Copper-free click chemistry systemsbased on activated alkynes circumvent toxic catalysts.

Activated alkynes often take the form of cyclooctynes, whereincorporation into the cyclooctyl group introduces ring strain to thealkyne.

Heteroatoms or substituents may he introduced at various locations inthe cyclooctyl ring, which may alter the reactivity of the alkyne orafford other alternative chemical properties in the compound. Variouslocations on the ring may also serve as attachment points for linkingthe cyclooctyne to a nucleic acid templated assembly moiety or linker.These locations on the ring or its substituents may optionally befurther derivatized with accessory groups.

Multiple cyclooctynes are commercially available, including severalderivatized versions suitable for use with standard bioconjugationchemistry protocols. Commercially available cyclooctyne derivatizednucleotides can aid in facilitating convenient incorporation of theligand during nucleic acid recognition moiety synthesis.

Cyclooctyne-azide based bio-orthogonal chemistry may produce templatedassembly products of the general structure:

Another example:

Azide-Phosphine Staudinger Chemistry

The Staudinger reduction, based on the rapid reaction between an azideand a phosphine or phosphite with loss of N₂, also represents abio-orthogonal reaction. The Staudinger ligation, in which covalentlinks are formed between the reactants in a Staudinger reaction, issuited for use in nucleic acid templated assembly. Both non-tracelessand traceless forms of the Staudinger ligation allow for a diversity ofoptions in the chemical structure of products formed in these reactions.

Non-Traceless Staudinger Ligation

The standard Staudinger ligation is a non-traceless reaction between anazide and a phenyl-substituted phosphine such as triphenylphosphine,where an electrophilic trap substituent on the phosphine, such as amethyl ester, rearranges with the aza-ylide intermediate of the reactionto produce a ligation product linked by a phosphine oxide. An example ofa Staudinger ligation product formed by haplomers A and B may have thestructure:

Phenyl-substituted phosphines carrying electrophilic traps can also bereadily synthesized. Derivatized versions are available commercially andsuitable for incorporation into haplomers:

Traceless Staudinger Ligation

In some embodiments, phosphines capable of traceless Staudingerligations may be utilized as bio-orthogonal moieties for ligands. In atraceless reaction, the phosphine serves as a leaving group duringrearrangement of the aza-ylide intermediate, creating a ligationtypically in the form of a native amide bond. Compounds capable oftraceless Staudinger ligation generally take the form of a thioesterderivatized phosphine or an ester derivatized phosphine:

An exemplary ester-derivatized phosphine for traceless Staudingerligation is:

An exemplary thioester-derivatized phosphine for traceless Staudingerligations is:

Chemical linkers or accessory groups may optionally be appended assubstituents to the R groups in the above structures, providingattachment points for nucleic acid recognition moieties or for theintroduction of additional functionality to the reactant.

Traceless Phosphinophenol Staudinger Ligation

Compared to the non-traceless Staudinger phenylphosphine compounds, theorientation of the electrophilic trap ester on a tracelessphosphinophenol is reversed relative to the phenyl group. This enablestraceless Staudinger ligations to occur in reactions with azides,generating a native amide bond in the product without inclusion of thephosphine oxide.

The traceless Staudinger ligation may be performed in aqueous mediawithout organic co-solvents if suitable hydrophilic groups, such astertiary amines, are appended to the phenylphosphine. Weisbrod and Marxdescribes preparation of water-soluble phosphinophenol, which may beloaded with a desired ligand containing a carboxylic acid (such as theC-terminus of a peptide) via the mild Steglich esterification using acarbodiimide such as dicyclohexylcarbodiimide (DCC) orN,N′-diisopropylcarbodiimide (DIC) and an ester-activating agent such as1-hydroxybenzotriazole (HOBT). This approach facilitates synthesis ofhaplomers of the form:

(Synlett, 2010, 5, 787-789).

Water-soluble phosphinophenol-based traceless haplomer structure.

Traceless Phosphinomethanethiol Staudinger Ligation

Phosphinomethanethiols represent an alternative to phosphinophenols formediating traceless Staudinger ligation reactions. In general,phosphinomethanethiols possess favorable reaction kinetics compared withphosphinophenols in mediating traceless Staudinger reaction. U.S. PatentApplication Publication 2010/0048866 and Tam et al., J. Am. Chem, Soc.,:2007, 129, 11421-30 describe preparation of water-solublephosphinomethanethiols of the form:

These compounds may be loaded with a peptide or other payload, in theform of an activated ester, to form a thioester suitable for use as atraceless bio-orthogonal reactive group:

Haplomer structure based on water-soluble phosphinomethanethioltraceless Staudinger bio-orthogonal chemistry.

Native Chemical Ligation

Native chemical ligation is a bio-orthogonal approach based on thereaction between a thioester and a compound bearing a thiol and an amineThe classic native chemical ligation is between a peptide bearing aC-terminal thioester and another bearing an N-terminal cysteine, as seenbelow:

Native chemical ligation may be utilized to mediate traceless reactionsproducing a peptide or peptidomimetic containing an internal cysteineresidue, or other thiol-containing residue if non-standard amino acidsare utilized.

N-terminal cysteines may be incorporated by standard amino acidsynthesis methods. Terminal thioesters may be generated by severalmethods known in the art, including condensation of activated esterswith thiols using agents such as dicyclohexylcarbodiimide (DCC), orintroduction during peptide synthesis via the use of “Safety-Catch”support resins.

Other Selectively Reactive Moieties

Any suitable bio-orthogonal reaction chemistry may be utilized forsynthesis of haplomer-ligand complexes, as long as it efficientlymediates a reaction in a highly selective manner in complex biologicenvironments. A recently developed non-limiting example of analternative bio-orthogonal chemistry that may be suitable is reactionbetween tetrazine and various alkenes such as norbornene andtrans-cyclooctene, which efficiently mediates bio-orthogonal reactionsin aqueous media.

Chemical linkers or accessory groups may optionally be appended assubstituents to the above structures, providing attachment points fornucleic acid recognition moieties or ligands, or for the introduction ofadditional functionality to the reactant.

The configurations involving the ligands depicted in the Examples andFigures could be reversed. In other words, the ligand could be linked tothe 3′ end of the bottle haplomer-ligand complex, as long as the secondhaplomer-ligand complex accordingly had its ligand linked to its 5′ end.The Examples provided below have the bottle haplomer-ligand complex witha 5′-linked ligand and the second haplomer-ligand complex with a3′-linked ligand. Likewise, in this system, the bio-orthogonal moietiescan be switched around. For example, instead of using the bottlehaplomer-ligand complex with a 5′-hexynyl and the second haplomer-ligandcomplex with a 3′-azide, the bottle haplomer-ligand complex could bearthe azide, and the second haplomer-ligand complex the hexynyl group.

In some embodiments, the bio-orthogonal moiety is chosen from an azide,an alkyne, a cyclooctyne, a nitron, a norbornene, an oxanorbomadiene, aphosphine, a dialkyl phosphine, trialkyl phosphine, a phosphinothiol, aphosphinophenol, a cyclooctene, a nitrile oxide, a thioester, atetrazine, an isonitrile, a tetrazole, or a quadricyclane, or anyderivative thereof. In some embodiments, the bio-orthogonal moiety ofthe first haplomer is hexynyl and the bio-orthogonal moiety of thesecond haplomer is azide. In some embodiments, the bio-orthogonal moietyof the first haplomer is azide and the bio-orthogonal moiety of thesecond haplomer is hexynyl.

For example, some embodiments use modifications of the FKM-NHS series ofcompounds (FIGS. 6-8 ) with click-group side chains. In someembodiments, FKM-PEG3-NHS is modified at the C2 position of the PEGchain with a methyltetrazine group (FKM-PEG3-MTZ-NHS) (see, FIG. 21 ),or a trans-cyclooctene group (FKM-PEG3-TCO-NHS) (see, FIG. 22 ). Whenthese compounds are appended to amino-labeled oligonucleotides viastandard NHS chemistry to form click-modified ligand haplomers (see,FIG. 20 ), they can react with each other in a bio-orthogonal fashionafter templating places them in close spatial proximity.FKM-PEG3-MTZ-NHS is:

where x is from 1 to 6; and FKM-PEG3-TCO-NHS is

where x is from 1 to 6.

In some embodiments, the ligand of one of the first haplomer-ligandcomplex or second haplomer-ligand complex is an FKM monovalent ligandthat is FKM-PEG3-MTZ-NHS and the ligand of the other of the firsthaplomer-ligand complex or second haplomer-ligand complex is an FKMmonovalent ligand that is FKM-PEG3-TCO-NHS. In some embodiments, theligand of one of the bottle haplomer-ligand complex and secondhaplomer-ligand complex is an FKM monovalent ligand that isFKM-PEG3-MTZ-NHS and the ligand of the other of the bottlehaplomer-ligand complex and second haplomer-ligand complex is an FKMmonovalent ligand that is FKM-PEG3-TCO-NHS.

The present disclosure also provides a compound having the formula:

where m is from 3 to 6.

The present disclosure also provides a compound having the formula:

where m is from 3 to 6.

The present disclosure also provides a compound having the formula:

where n is from 1 to 6.

The present disclosure also provides a compound having the formula:

where x is from 1 to 6.

The present disclosure also provides a compound having the formula:

where x is from 1 to 6.

The present disclosure also provides a compound having the formula:

which is MFL2.

The present disclosure also provides fusion proteins comprising afragment of a protein of interest fused to a ligand binding domain,wherein the ligand binding domain is a ligand binding domain for smallmolecule ligands, or the ligand binding domain is an interactive proteindomain.

In some embodiments, the ligand binding domain is a ligand bindingdomain for small molecule ligands. In some embodiments, the ligandbinding domain is an FKBP domain or an FRB domain. In some embodiments,the FKBP domain is a mutant FKBP domain. In some embodiments, the mutantFKBP domain is the F36V FKBP mutant domain comprising the amino acidsequence GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATINFDVELL KLE (SEQ IDNO:14) or MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPITATLVFD VELLKLE(SEQ ID NO:15).

In some embodiments, the ligand binding domain is an interactive proteindomain. In some embodiments, the interactive protein domain comprisesless than 100 amino acid residues. In some embodiments, the interactiveprotein domain is a leucine sipper domain. In some embodiments, theinteractive protein domain is a c-jun domain, a c-fos domain, a c-mycdomain, a c-max domain, an NZ domain, or a CZ domain.

In some embodiments, the interactive protein domain fused to theN-terminus of the protein of interest. In some embodiments, the,interactive protein domain is fused to the C-terminus of the protein ofinterest.

In some embodiments, the NZ domain comprises the amino acid sequenceALKKELQANKKELAQLKWELQALKKELAQ (SEQ ID NO:10), and the CZ domaincomprises the amino acid sequence EQLEKKLQALEKKLAQLEWKNQALEKKLAQ (SEQNO:11).

In some embodiments, the c-jun domain comprises the amino acid sequence

(SEQ ID NO: 1) GGASLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKGAP(SEQ ID NO: 4) GGASLERIARLEEKVKSFKAQNSENASTANMLREQVAQLKQKGAP(SEQ ID NO: 16) ASLERIARLEEKVKSFKAQNSENASTANMLREQVAQLKQKGAP.

In some embodiments, the c-jun domain comprises the amino acid sequence

(SEQ ID NO: 2) SGASLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKGAPSGGC or(SEQ ID NO: 5) SGASLERIARLEEKVKSFKAQNSENASTANMLREQVAQLKQKGAPSGGC.

In some embodiments, the c-Fos domain comprises the amino acid sequence

(SEQ ID NO: 3) ASRELTDTLQAETDQLEDEKSALQTEIANLLKEKEKLEGAP or(SEQ ID NO: 13) SGASRELTDTLQAETDQLEDEKSALQTEIANLLKEKEKLEGAP.

In some embodiments, the fusion protein comprises a linker between theprotein of interest and the ligand binding domain. In some embodiments,the linker is a Ser/Gly linker, a Poly-Asparagine linker, or a linkercomprising the amino acid sequence AGSSAAGSGS (SEQ ID NO:17). In someembodiments, the Poly-Asparagine linker comprises from about 8 to about16 asparagine residues. In some embodiments, the Ser/Gly linkercomprises GGSGGGSGGGS GGGSGGG (SEQ ID NO:18), GGSGGGSGGGSGGGSGGGSGGG(SEQ ID NO:19), GGSGGGSGGGSGGGSGGGSGGGSGGG (SEQ ID NO:20),SGGGGSGGGGSGGGG (SEQ ID NO:21), SGGGGSGGGGSGGGGSGGGG (SEQ ID NO:22),SGGGGSGGGGSGGGGSGG GGSGGGG (SEQ ID NO:23), SGGGS (SEQ ID NO:24), SGSG(SEQ ID NO:25), SGGGGS (SEQ ID NO:26), or SGSGG (SEQ ID NO:27).

The protein of interest, whose fragments are brought together via theirrespective fusion to the respective ligand binding domain, can be anydesired protein capabale of folding together or dimerization.

In some embodiments, the protein of interest may trigger activity byacting within a target compartment (for example, within a cell), at thesurface of a target compartment (for example, at the cell surface), inthe vicinity of the target compartment (for example, when the effectorstructure is actively exported from the cell, leaks from the cell, orreleased upon cell death), or diffuse or be carried to a distant regionof the sample to trigger a response. In some embodiments, the protein ofinterest can be targeted to their active sites by incorporation oftargeting groups in the templated assembly product. Examples oftargeting groups include, but are not limited to, endoplasmic reticulumtransport signals, golgi apparatus transport signals, nuclear transportsignals, mitochondrial transport signals, ubiquitination motifs, otherproteosome targeting motifs, or glycosylphosphatidylinositol anchormotifs. Targeting groups may be introduced by their incorporation into ahaplomer moiety, chemical linker, or accessory group during synthesis,or may be generated during the ligation reaction.

In some embodiments, the protein of interest can be presented on thesurface of a target compartment. In some embodiments, the protein ofinterest can be presented on the surface of a cell as a ligand bound toa major histocompatibility complex molecule.

In some embodiments, the protein of interest can be an endogenouspeptide, and their analogue, or a completely synthetic structure whichis a target for agents such as antibodies. Because the availability oftarget nucleic acid molecules can limit production of active proteins ofinterest, it may be desirable to have proteins of interest that exertactivity when present at low levels.

In some embodiments, killing or growth inhibition of target cells can beinduced by direct interaction with cytotoxic, microbicidal, or virucidaleffector structures. Numerous toxic molecules known in the art can beproduced. In some embodiments, the protein of interest is a toxicpeptide. Examples of toxic peptides include, but are not limited to, beemelittin, conotoxin, cathelicidins, defensins, protegrins, and NK-lysin.

In some embodiments, killing or growth inhibition of target cells can beinduced by pro-apoptotic proteins of interest. For example, proteins ofinterest include pro-apoptotic peptides, including but not limited to,prion protein fragment 106-126 (PrP 106-126), Bax-derived minimumporopeptides associated with the caspase cascade including Bax 106-134,and pro-apoptotic peptide (KLAKLAKKLAKLAK; SECS ID NO:28).

In some embodiments, the protein of interest can be thrombogenic, inthat it induces activation of various components of the clotting cascadeof proteins, or activation of proteins, or activation and/or aggregationof platelets, or endothelial damage that can lead to a biologicallyactive process in which a region containing pathogenic cells can beselectively thrombosed to limit the blood supply to a tumor or otherpathogenic cell. These types of proteins of interest can also induceclotting, or prevent clotting, or prevent platelet activation andaggregation in and around targeted pathogenic cells.

In some embodiments, proteins of interest can mediate killing or growthinhibition of target cells or viruses by activating molecules, pathways,or cells associated with the immune system. Proteins of interest mayengage the innate immune system, the adaptive immune system, and/orboth.

In some embodiments, proteins of interest can mediate killing or growthinhibition of cells or viruses by stimulation of the innate immunesystem. In some embodiments, proteins of interest includepathogen-associated molecular patterns (PAMPs), damage-associatedmolecular patterns (DAMPs), and synthetic analogues thereof.

In some embodiments, the innate immune system can be engaged by proteinsof interest that activate the complement system. A non-limiting exampleof a complement activating effector structures can be the C3a fragmentof complement protein C3.

In some embodiments, proteins of interest can be natural or syntheticligands of Toll-Like Receptors (TLR). Examples of such proteins ofinterest include peptide fragments of heat shock proteins (hsp) known tobe TLR agonists.

In some embodiments, traceless bio-orthogonal chemistry may be used toproduce the muramyl dipeptide agonist of the NOD2 receptor to activatean inflammatory response.

In some embodiments, proteins of interest can mediate killing or growthinhibition of cells or viruses by activating molecules or cells of theadaptive immune system. Unique to the adaptive immune system, moleculesor cells can be engineered to recognize an extraordinary variety ofstructures, thus removing the constraint that the proteins of interestmust be intrinsically active or bind to an endogenous protein.

In some embodiments, proteins of interest can be a ligand for anantibody or antibody fragment (including but not limited to Fab, Fv, andscFv). Traceless bio-orthogonal approaches can be used to produce apeptide or other epitope that is bound by an existing antibody, or anantibody can be developed to recognize proteins of interest created.

In some embodiments, the protein of interest is a fragment of: acytotoxic protein, a microbicidal protein, a virucidal protein, apro-apoptotic protein, a thrombogenic protein, a complement activatingprotein, a Toll-Like Receptor protein, a NOD2 receptor agonist protein,or an antibody or fragment thereof, wherein the first fragment and thesecond fragment dimerize or fold together.

In some embodiments, the cytotoxic protein is a bee melittin, aconotoxin, a cathelicidin, a defensin, a protegrin, or NK-lysin. In someembodiments, the pro-apoptotic protein is prion protein, a Bax-derivedminimum poropeptide associated with the caspase cascade, or apro-apoptotic peptide (KLAKLAKKLAKLAK) (SEQ ID NO:28). In someembodiments, the innate immune system stimulation protein is apathogen-associated molecular pattern (PAMP) or a damage-associatedmolecular pattern (DAMP). In some embodiments, the complement activatingprotein is a C3a fragment of complement protein C3. In some embodiments,the Toll-Like Receptor (TLR) protein is a heat shock protein (hsp). Insome embodiments, the NOD2 receptor agonist protein is muramyl dipeptideagonist. In some embodiments, the antibody fragment is an Fab, Fv, orscFv.

In some embodiments, the protein of interest is a fragment of:superfolder GFP (sfGFP), Renilla luciferase, murine dihydrofolatereductase (DHFR), S. cerevisiae ubiquitin, β-lactamase, or Herpessimplex virus type 1 thymidine kinase, wherein one fragment of theprotein of interest dimerizes or folds together with the other fragmentof the protein of interest.

In some embodiments, the fragment of superfolder GFP (sfGFP) comprisesMRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDONILOHKLEYNFNSHNVYITADKQ (SEQ ID NO:29) or KNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK (SEQ ID NO:30), wherein one fragment dimerizes or foldstogether with the other fragment.

In some embodiments, the fragment of Renilla luciferase comprisesMASKVYDPEQR KRMITGPQWWARCKQMNVLDSFINYYDSEKHAENAVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKS GKS GNGSYRLLDHYKY LTAWFELLNLPKKHFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDIALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPREIPLVKGG (SEQ ID NO:31) orKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKVKGLHFSQEDAPDEMGKYIKSFVERVLKNEQ (SEQ ID NO:32), wherein one fragment dimerizesor folds together with the other fragment.

In some embodiments, the fragment of murine dihydrofolate reductase(DHFR) comprises amino acids 1-105 or 106-186 thereof, wherein onefragment dimerizes or folds together with the other fragment.

In some embodiments, the fragment of S. cerevisiae ubiquitin comprisesamino acids 1-34 (MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKE; SEQ ID NO:33) or35-76 (GIPPDQQ RLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG; SEQ ID NO:34)thereof, wherein one fragment dimerizes or folds together with the otherfragment.

In some embodiments, the fragment of β-lactamase comprises amino acids25-197 or 198-286 thereof, wherein one fragment dimerizes or foldstogether with the other fragment.

In some embodiments, the fragment of Herpes simplex virus type 1thymidine kinase comprises amino acids 1-265 or 266-376 thereof, whereinone fragment dimerizes or folds together with the other fragment.

In some embodiments, there may be no pre-existing information regardingwhere a protein of interest may be divided for general split-proteinanalyses, including LD-TAPER. In such cases, inspection of thethree-dimensional crystal structure of the protein may provide a numberof candidate targets within surface loops and turns, away from regionsdirectly concerned with the protein's function. Fragments arising fromcleavage at a predicted target site may be screened by separateexpression as fusion proteins with suitable mutually interactive leucinezippers, where protein activity is restored upon mixing of fusionproteins if the split protein targeting is successful. More rapid assaysfor empirically flagging suitable cleavage sites are available,including solubility assays (see, Chen et al., Protein Science, 2009,18, 399-409), or the preferred circular permutation assay (see, Massoudet al., Nature Medicine, 2010, 16, 921-926). These assays are applicableeven in the absence of structural information, but can be guided andmade more efficient by structural knowledge where available. For thecircular permutation assay, a tandem in-frame continuous dimer of thecoding sequence of interest is initially generated, with aserine-glycine linker (such as SGGGGSGGGGSGGGG; SEQ ID NO:21) positionedbetween the two copies. Circularly permuted coding sequence blocks forexpression are then generated from the dimer by amplification usingsuitable primers.

In some embodiments, the fragment of the protein of interest of a firstfusion protein and the fragment of the protein of interest of a secondfusion protein can dimerize or fold together. In some embodiments, afirst fusion protein comprises a fragment of a protein of interest fusedto a ligand binding domain for a small molecule ligand, and a secondfusion protein comprises a fragment of a protein of interest fused to aligand binding domain for a small molecule ligand. In some embodiments,a first fusion protein comprises a fragment of a protein of interestfused to an interactive protein domain, and a second fusion proteincomprises a fragment of a protein of interest fused to an interactiveprotein domain.

In some embodiments, the interactive protein domain of a first fusionprotein is fused to the N-terminus of the fragment of the protein ofinterest, and the interactive protein domain of a second fusion proteinis fused to the N-terminus of the fragment of the protein of interest;or the interactive protein domain of a first fusion protein is fused tothe C-terminus of the fragment of the protein of interest, and theinteractive protein domain of a second fusion protein is fused to theC-terminus of the fragment of the protein of interest; or theinteractive protein domain of one of the first fusion protein and secondfusion protein is fused to the N-terminus of the fragment of the proteinof interest, and the interactive protein domain of the other of thefirst fusion protein and second protein is fused to the C-terminus offragment of the protein of interest.

In some embodiments, both the first fusion protein and second fusionprotein comprise a linker between the protein of interest and the ligandbinding domain. In some embodiments, each linker is, independently, aSer/Gly linker, a Poly-Asparagine linker, or a linker comprising theamino acid sequence AGSSAAGSGS (SEQ ID NO:17), as described herein.

The present disclosure also provides compositions or kits comprising anyone or more of: the haplomer-ligand complexes, the bottlehaplomer-ligand complexes, and the fusion proteins described herein.

In some embodiments, the composition or kit comprises: a firsthaplomer-ligand complex described herein comprising a small moleculeligand; a second haplomer-ligand complex described herein comprising asmall molecule ligand; a first fusion protein comprising a fragment of aprotein of interest fused to a ligand binding domain for a smallmolecule ligand; and a second fusion protein comprising a fragment of aprotein of interest fused to a ligand binding domain for a smallmolecule ligand; wherein: i) the ligand of the first haplomer-ligandcomplex is linked to the 5′ terminus of the polynucleotide of the firsthaplomer-ligand complex; ii) the ligand of the second haplomer-ligandcomplex is linked to the 3′ terminus of the polynucleoitide of thesecond haplomer-ligand complex; iii) the polynucleotide of the firsthaplomer-ligand complex is substantially complementary to a targetnucleic acid molecule; iv) the polynucleotide of the secondhaplomer-ligand complex is substantially complementary to the targetnucleic acid molecule at a site in spatial proximity to thepolynucleotide of the first haplomer-ligand complex; v) the ligand ofthe first haplomer-ligand complex and the ligand binding domain of thefirst fusion protein can interact; vi) the ligand of the secondhaplomer-ligand complex and the ligand binding domain of the secondfusion protein can interact; and vii) the fragment of the protein ofinterest of the first fusion protein and the fragment of the protein ofinterest of the second fusion protein can dimerize or fold together.

In some embodiments, the composition or kit comprises: a firsthaplomer-ligand complex comprising of a ligand that is all interactiveprotein domain; a second haplomer-ligand complex comprising of a ligandthat is an interactive protein domain; a first fusion protein comprisinga fragment of a protein of interest fused to an interactive proteindomain; and a second fusion protein comprising a fragment of a proteinof interest fused to an interactive protein domain; wherein: i) theligand of the first haplomer-ligand complex is linked to the 5′ terminusof the, polynucleoitide of the first haplomer-ligand complex; ii) theligand of the second haplomer-ligand complex is linked to the 3′terminus of the polynucleotide of the second haplomer-ligand complex;iii) the polynucleotide of the first haplomer-ligand complex issubstantially complementary to a target nucleic acid molecule; iv) thepolynucleotide of the second haplomer-ligand complex is substantiallycomplementary to the target nucleic acid molecule at a site in spatialproximity to the polynucleotide of the first haplomer-ligand complex; v)the ligand of the first haplomer-ligand complex and the ligand bindingdomain of the first fusion protein can interact; vi) the ligand of thesecond haplomer-ligand complex and the ligand binding domain of thesecond fusion protein can interact; and vii) the fragment of the proteinof interest of the first fusion protein and the fragment of the proteinof interest of the second fusion protein can dimerize or fold together.

In some embodiments, the composition or kit comprises: a first bottlehaplomer-ligand complex as described herein comprising a small moleculeligand; a second haplomer-ligand complex described herein comprising asmall molecule ligand, wherein the second haplomer-ligand complexcomprises a nucleotide portion that is substantially complementary tothe stem portion of the bottle haplomer-ligand complex that is linked tothe ligand of the bottle haplomer-ligand complex; a first fusion proteina fragment of a protein of interest fused to a ligand binding domain fora small molecule ligand; and a second fusion protein a fragment of aprotein of interest fused to a ligand binding domain for a smallmolecule ligand; wherein: i) the ligand of the first bottlehaplomer-ligand complex and the ligand binding domain of the firstfusion protein can interact; ii) the ligand of the secondhaplomer-ligand complex and the ligand binding domain of the secondfusion protein can interact; and iii) the fragment of the protein ofinterest of the first fusion protein and the fragment of the protein ofinterest of the second fusion protein can dimerize or fold together.

In some embodiments, the composition or kit comprises: a first bottlehaplomer-ligand complex described herein comprising a ligand that is aninteractive protein domain; second haplomer-ligand complex comprising ofa ligand that is an interactive protein domain, wherein the secondhaplomer-ligand complex comprises a nucleotide portion that issubstantially complementary to the stem portion of the bottlehaplomer-ligand complex that is linked to the ligand of the bottlehaplomer-ligand complex; a first fusion protein comprising a fragment ofa protein of interest fused to an interactive protein domain; and asecond fusion protein comprising a fragment of a protein of interestfused to an interactive protein domain; wherein: i) the ligand of thefirst bottle haplomer-ligand complex and the ligand binding domain ofthe first fusion protein can interact; ii) the ligand of the secondhaplomer-ligand complex and the ligand binding domain of the secondfusion protein can interact; and iii) the fragment of the protein ofinterest of the first fusion protein and the fragment of the protein ofinterest of the second fusion protein can dimerize or fold together.

The present disclosure also provides methods of using the any of thehaplomer-ligand complexes described herein in combination with thefusion proteins described herein to produce a dimerized or foldedprotein.

In some embodiments, the method comprises: a) contacting a targetnucleic acid molecule with a first haplomer-ligand complex comprising asmall molecule ligand; b) contacting the target nucleic acid with asecond haplomer-ligand complex comprising a small molecule ligand; c)contacting the first haplomer-ligand complex with a first fusion proteinthat comprises a ligand binding domain for a small molecule ligand; andd) contacting the second haplomer-ligand complex with a second fusionprotein that comprises a ligand binding domain for a small moleculeligand; wherein: i) the ligand of the first haplomer-ligand complex islinked to the 5′ terminus of the polynucleotide of the firsthaplomer-ligand complex; ii) the ligand of the second haplomer-ligandcomplex is linked to the 3′ terminus of the polynucleotide of the secondhaplomer-ligand complex; iii) the polynucleotide of the firsthaplomer-ligand complex is substantially complementary to a targetnucleic acid molecule; iv) the polynucleotide of the secondhaplomer-ligand complex is substantially complementary to the targetnucleic acid molecule at a site in spatial proximity to thepolynucleotide of the first haplomer-ligand complex; v) the ligand ofthe first haplomer-ligand complex and the ligand binding domain of thefirst fusion protein can interact; and vi) the ligand of the secondhaplomer-ligand complex and the ligand binding domain of the secondfusion protein can interact; thereby resulting in the folding ordimerization of the fragment of the protein of interest of the firstfusion protein with the fragment of the protein of interest of thesecond fusion protein.

In some embodiments, the method comprises: a) contacting a targetnucleic acid molecule with a first haplomer-ligand complex comprising aligand that is an interactive protein domain; b) contacting the targetnucleic acid with a second haplomer-ligand complex comprising a ligandthat is an interactive protein domain; c) contacting the firsthaplomer-ligand complex with a first fusion protein that comprises afragment of a protein of interest fused to an interactive proteindomain; and d) contacting the second haplomer-ligand complex with asecond fusion protein that comprises a fragment of a protein of interestfused to an interactive protein domain; wherein: i) the ligand of thefirst haplomer-ligand complex is linked to the 5′ terminus of thepolynucleotide of the first haplomer-ligand complex; ii) the ligand ofthe second haplomer-ligand complex is linked to the 3′ terminus of thepolynucleotide of the second haplomer-ligand complex; iii) thepolynucleotide of the first haplomer-ligand complex is substantiallycomplementary to a target nucleic acid molecule; iv) the polynucleotideof the second haplomer-ligand complex is substantially complementary tothe target nucleic acid molecule at a site in spatial proximity to thepolynucleotide of the first haplomer-ligand complex; v) the ligand ofthe first haplomer-ligand complex and the ligand binding domain of thefirst fusion protein can interact; and vi) the ligand of the secondhaplomer-ligand complex and the ligand binding domain of the secondfusion protein can interact; thereby resulting in the folding ordimerization of the fragment of the protein of interest of the firstfusion protein with the fragment of the protein of interest of thesecond fusion protein.

In some embodiments, the polynucleotide of the first haplomer-ligandcomplex is complementary to the polynucleotide of the secondhaplomer-ligand complex. In some embodiments, the polynucleotide of thefirst haplomer-ligand complex binds to the target nucleic acid moleculein spatial proximity to the binding of the polynucleotide of the secondhaplomer-ligand complex to the target nucleic acid molecule. In someembodiments, the ligand of the first haplomer-ligand complex is linkedto the 5′ terminus of the polynucleotide of the first haplomer-ligandcomplex, and the polynucleotide of the first haplomer-ligand complex iscomplementary to a portion of the nucleic acid target 5′ adjacent to astem-loop structure; and the ligand of the second haplomer-ligandcomplex is linked to the 3′ terminus of the polynucleotide of the secondhaplomer-ligand complex, and the polynucleotide of the secondhaplomer-ligand complex is complementary to a portion of the nucleicacid target 3′ adjacent to the stem-loop structure. In some embodiments,the ligand of the first haplomer-ligand complex is linked to the 3′terminus of the polynucleotide of the first haplomer-ligand complex, andthe polynucleotide of the first haplomer-ligand complex is complementaryto a 5′ portion of a loop structure of a stem-loop structure of thenucleic acid target, wherein the 5′ portion of the loop structure isadjacent to the stem region of the stem-loop structure; and the ligandof the second haplomer-ligand complex is linked to the 5′ terminus ofthe polynucleotide of the second haplomer-ligand complex, and thepolynucleotide of the second haplomer-ligand complex is complementary toa 3′ portion of the loop structure of the stem-loop structure of thenucleic acid target, wherein the 3′ portion of the loop structure isadjacent to the stem region of the stem-loop structure.

In some embodiments, the method comprises: a) contacting a targetnucleic acid molecule with a complex formed by the interaction of afirst haplomer-ligand complex comprising a small molecule ligand with afirst fusion protein that comprises a ligand binding domain for a smallmolecule ligand, wherein the ligand of the first haplomer-ligand complexis linked to the 5′ terminus of the first haplomer-ligand complex, andwherein the ligand of the first haplomer-ligand complex interacts withthe ligand binding domain of the first fusion protein; and b) contactingthe target nucleic acid molecule with a complex formed by theinteraction of a second haplomer-ligand complex comprising a smallmolecule ligand with a second fusion protein that comprises a ligandbinding domain for a small molecule ligand, wherein the ligand of thesecond haplomer-ligand complex is linked to the 5′ terminus of thepolynucleotide of the second haplomer-ligand complex, and wherein theligand of the second haplomer-ligand complex interacts with the ligandbinding domain of the second fusion protein; thereby resulting in thefolding or dimerization of the fragment of the protein of interest ofthe first fusion protein with the fragment of the protein of interest ofthe second fusion protein.

In some embodiments, the method comprises: a) contacting a targetnucleic acid molecule with a complex formed by the interaction of afirst haplomer-ligand complex comprising a ligand that is an interactiveprotein domain with a first fusion protein that comprises a fragment ofa protein of interest fused to an interactive protein domain, whereinthe ligand of the haplomer-ligand complex is linked to the 5′ terminusof the polynucleotide of the first, haplomer-ligand complex, and whereinthe ligand of the first haplomer-ligand complex interacts with theligand binding domain of the first fusion protein; and b) contacting thetarget nucleic acid molecule with a complex thrilled by the interactionof a second haplomer-ligand complex comprising a ligand that is aninteractive protein domain with a second fusion protein that comprises afragment of a protein of interest fused to an interactive proteindomain, wherein the ligand of the second haplomer-ligand complex islinked to the 5′ terminus of the polynucleotide of the secondhaplomer-ligand complex, and wherein the ligand of the secondhaplomer-ligand complex interacts with the ligand binding domain of thesecond fusion protein; thereby resulting in the folding or dimerizationof the fragment of the protein of interest of the first fusion proteinwith the fragment of the protein of interest of the second fusionprotein.

In some embodiments, the polynucleotide of the first haplomer-ligandcomplex is complementary to the polynucleotide of the secondhaplomer-ligand complex. In some embodiments, the polynucleotide of thefirst haplomer-ligand complex binds to the target nucleic acid moleculein spatial proximity to the binding of the polynucleotide of the secondhaplomer-ligand complex to the target nucleic acid molecule. In someembodiments, the ligand of the first haplomer-ligand complex is linkedto the 5′ terminus of the polynucleotide of the first haplomer-ligandcomplex, and the polynucleotide of the first haplomer-ligand complex iscomplementary to a portion of the nucleic acid target 5′ adjacent to astem-loop structure; and the ligand of the second haplomer-ligandcomplex is linked to the 3′ terminus of the polynucleotide of the secondhaplomer-ligand complex, and the polynucleotide of the secondhaplomer-ligand complex is complementary to a portion of the nucleicacid target 3′ adjacent to the stem-loop structure. In some embodiments,the ligand of the first haplomer-ligand complex is linked to the 3′terminus of the polynucleotide of the first haplomer-ligand complex, andthe polynucleotide of the first haplomer-ligand complex is complementaryto a 5′ portion of a loop structure of a stem-loop structure of thenucleic acid target, wherein the 5′ portion of the loop structure isadjacent to the stem region of the stem-loop structure; and the ligandof the second haplomer-ligand complex is linked to the 5′ terminus ofthe polynucleotide of the second haplomer-ligand complex, and thepolynucleotide of the second haplomer-ligand complex is complementary toa 3′ portion of the loop structure of the stem-loop structure of thenucleic acid target, wherein the 3′ portion of the loop structure isadjacent to the stem region of the stem-loop structure.

In some embodiments, the method comprises: a) contacting a targetnucleic acid molecule with a bottle haplomer-ligand complex describedherein comprising a small molecule ligand; b) contacting the targetnucleic acid with a second haplomer-ligand complex comprising a smallmolecule ligand, wherein the second haplomer-ligand complex comprises anucleotide portion that is substantially complementary to the stemportion of the bottle haplomer-ligand complex that is linked to theligand of the bottle haplomer-ligand complex; c) contacting the bottlehaplomer-ligand complex with a first fusion protein that comprises aligand binding domain for a small molecule ligand, wherein the ligand ofthe bottle haplomer-ligand complex and the ligand binding domain of thefirst fusion protein can interact; and d) contacting the secondhaplomer-ligand complex with a second fusion protein that comprises aligand binding domain for a small molecule ligand, wherein the ligand ofthe second haplomer-ligand complex and the ligand binding domain of thesecond fusion protein can interact; thereby resulting in the folding ordimerization of the fragment of the protein of interest of the firstfusion protein with the fragment of the protein of interest of thesecond fusion protein.

In some embodiments, the method comprises: a) contacting a targetnucleic acid molecule with a bottle haplomer-ligand complex comprising aligand that is an interactive protein domain; b) contacting the targetnucleic acid with a second, haplomer-ligand complex comprising a ligandthat is all interactive protein domain, wherein the secondhaplomer-ligand complex comprises a nucleotide portion that issubstantially complementary to the stem portion of the bottlehaplomer-ligand complex that is linked to the ligand of the bottlehaplomer-ligand complex; c) contacting the bottle haplomer-ligandcomplex with a first fusion protein that comprises a fragment of aprotein of interest fused to an interactive protein domain, wherein theligand of the bottle haplomer-ligand complex and the ligand bindingdomain of the first fusion protein can interact; and d) contacting thesecond haplomer-ligand complex with a second fusion protein thatcomprises a fragment of a protein of interest fused to an interactiveprotein domain, wherein the ligand of the second haplomer-ligand complexand the ligand binding domain of the second fusion protein can interact;thereby resulting in the folding or dimerization of the fragment of theprotein of interest of the first fusion protein with the fragment of theprotein of interest of the second fusion protein.

In some embodiments, the method comprises: a) contacting a targetnucleic acid molecule with a bottle haplomer-ligand complex comprising asmall molecule ligand; b) contacting the target nucleic acid moleculewith a second haplomer-ligand complex comprising a small moleculeligand, wherein the second haplomer-ligand complex comprises anucleotide portion that is substantially complementary to the stemportion of the bottle haplomer-ligand complex that is linked to theligand of the bottle haplomer-ligand complex; c) contacting the bottlehaplomer-ligand complex with a first fusion protein that comprises aligand binding domain for a small molecule ligand, wherein the ligand ofthe bottle haplomer-ligand complex and the ligand binding domain of thefirst fusion protein can interact; and d) contacting the secondhaplomer-ligand complex with a second fusion protein that comprises aligand binding domain for a small molecule ligand, wherein the ligand ofthe second haplomer-ligand complex and the ligand binding domain of thesecond fusion protein can interact; thereby resulting in the folding ordimerization of the fragment of the protein of interest of the firstfusion protein with the fragment of the protein of interest of thesecond fusion protein.

In some embodiments, the method comprises: a) contacting a targetnucleic acid molecule with a bottle haplomer-ligand complex comprising aligand that is an interactive protein domain; b) contacting the targetnucleic acid molecule with a second haplomer-ligand complex comprising aligand that is an interactive protein domain, wherein the secondhaplomer-ligand complex comprises a nucleotide portion that issubstantially complementary to the stem portion of the bottlehaplomer-ligand complex that is linked to the ligand of the bottlehaplomer-ligand complex; c) contacting the bottle haplomer-ligandcomplex with a first fusion protein that comprises a fragment of aprotein of interest fused to an interactive protein domain, wherein theligand of the bottle haplomer-ligand complex and the ligand bindingdomain of the first fusion protein can interact; and d) contacting thesecond haplomer-ligand complex with a second fusion protein thatcomprises a fragment of a protein of interest fused to an interactiveprotein domain, wherein the ligand of the second haplomer-ligand complexand the ligand binding domain of the second fusion protein can interact;thereby resulting in the folding or dimerization of the fragment of theprotein of interest of the first fusion protein with the fragment of theprotein of interest of the second fusion protein.

The present disclosure also provides methods of using any of thehaplomer-ligand complexes, bottle haplomer-ligand complexes, and fusionproteins described herein to modulate a cell or cell target molecule.Administration of sets of corresponding haplomer-ligand complexes alongwith their corresponding fusion proteins to a mammal, or to a human, mayvary according to the nature of the disease, disorder or conditionsought to be treated. In some embodiments, the haplomer-ligandcomplexes, bottle haplomer-ligand complexes, and fusion proteins can bedispensed into a sample within a suitable vessel or chamber. In someembodiments, the haplomer-ligand complexes, bottle haplomer-ligandcomplexes, and fusion proteins can be dispensed into a sample within asuitable vessel or chamber. In some embodiments, the sample may bedispensed a vessel already containing the haplomer-ligand complexes,bottle haplomer-ligand complexes, and fusion proteins. In someembodiments, the haplomer-ligand complexes, bottle haplomer-ligandcomplexes, and fusion proteins can used in vitro or in situ. In someembodiment, the human will be in need of such treatment.

In some embodiments, the haplomer-ligand complexes, bottlehaplomer-ligand complexes, and fusion proteins can be administered fortemplated assembly in vivo. To facilitate such treatment, preparedhaplomer-ligand complexes, bottle haplomer-ligand complexes, and fusionproteins may be administered in any suitable buffer or formulation,optionally incorporating a suitable delivery agent, and contacted withthe mammal or human, or sample thereof for ex vivo methods. Concentratedforms of haplomer-ligand complexes, bottle haplomer-ligand complexes,and fusion proteins may be handled separate from its counterparthaplomer-ligand complexes, bottle haplomer-ligand complexes, and fusionproteins, as product-generating reactions may occur in the absence oftarget nucleic acid molecule template at high concentrations. Table 1provides guidelines for maximum acceptable concentrations of gymnotic(no delivery agent) haplomers comprised of various haplomer-ligandcomplexes, bottle haplomer-ligand complexes, and fusion proteins. If thehaplomer-ligand complexes, bottle haplomer-ligand complexes, and fusionproteins are contacted at concentrations above these thresholds,non-templated background reactions may occur.

TABLE 1 Maximum concentrations for contact of haplomers, above whichnon-templated reaction levels may occur Bioorthogonal Reactive MaximumChemistry Concentration Azide-Alkyne <50 μm Azide-Phosphine <50 μmNative Chemical Ligation  <1 mM

Threshold concentrations of other haplomer-ligand complexes, bottlehaplomer-ligand complexes, and fusion proteins may be determinedempirically utilizing the templated assembly diagnostic evaluation assaydisclosed.

In some embodiments, the likelihood of non-templated reactions may bereduced by administering a set of corresponding haplomer-ligandcomplexes, bottle haplomer-ligand complexes, and fusion proteins suchthat one haplomer-ligand complex is administered first, then a timedelay is observed before the corresponding haplomer-ligand complex isadministered. This time delay may range from one minute to days,depending on the persistence of the haplomer-ligand complex in thesystem.

Certain delivery agents, such as transfection reagents such as cationiclipids, polyethyleneimine, dextran-based transfectants, or others knownin the art, may cause condensation of the haplomer-ligand complex. Underthese circumstances, haplomer-ligand complex may be prepared separatefrom the corresponding reactive haplomer-ligand complex and administeredto the sample separately. haplomer-ligand complex may also beadministered gymnotically, dissolved in an appropriate buffer withoutaddition of any additional delivery agent.

The haplomer-ligand complexes, bottle haplomer-ligand complexes, andfusion proteins may also he administered after formulation with asuitable delivery agent. A suitable delivery agent may enhance thestability, bioavailability, biodistribution, cell permeability, or otherdesirable pharmacologic property of the haplomer-ligand complexes,bottle haplomer-ligand complexes, and fusion proteins, or a combinationof these properties. Delivery agents known in the art include, but arenot limited to, polycationic transfection reagents, polyethyleneimineand its derivatives, DEAE-Dextran, other transfection reagents, salts,ions, buffers, solubilization agents, various viral vectors, liposomes,targeted liposomes, nanoparticles, carrier polymers, endosomedisruptors, permeabilization agents, lipids, steroids, surfactants,dispersants, stabilizers, or any combination thereof.

Delivery of haplomer-ligand complexes, bottle haplomer-ligand complexes,and fusion proteins to target compartments may also be enhanced bycovalent attachment of accessory groups to haplomer-ligand complexes,bottle haplomer-ligand complexes, and fusion proteins. Accessory groupsthat may enhance delivery may include compounds known to enhance thestability and biodistribution of compounds, such as polyethylene glycol(PEG); and enhance cell permeability of haplomers, including, but notlimited to, cholesterol derivatives known in the art,endosome-disrupting agents known in the art, and cell-penetratingpeptides, such as poly-cations such as poly-arginine or polylysine,peptides derived from the HIV tat protein, transportan, and peptidesderived from the antennapedia protein (penetratin).

Administration of effector protein product-triggered agents, such as anantibody or other effector protein product-detecting molecule, oreffector protein product-detecting cell, may also be included. Theadministration can be part of the templated assembly procedure. It maybe administered before, during, or after administration of thehaplomer-ligand complexes, bottle haplomer-ligand complexes, and fusionproteins, and by any method appropriate to the agent. In someembodiments, the effector protein product-triggered agent isadministered prior to administration of the haplomer-ligand complexes,bottle haplomer-ligand complexes, and fusion proteins to facilitatetriggering of activity by effector proteins as soon as they are formedand available for agent binding.

In some embodiments, multiple sets of corresponding haplomer-ligandcomplexes, bottle haplomer-ligand complexes, and fusion proteins may beadministered in parallel. These sets of reactants may bind to multiplehybridization sites on a single target nucleic acid, or bind todifferent target nucleic acids, or a combination thereof. The differentsets of haplomer-ligand complexes, bottle haplomer-ligand complexes, andfusion proteins may produce the same protein structure, thus increasingthe level of activity generated by that protein structure by boostingits production, or the different sets of haplomer-ligand complexes,bottle haplomer-ligand complexes, and fusion proteins may producedifferent protein structures, thus producing multivalent activity in thesample, or a combination thereof. When multiple sets of haplomer-ligandcomplexes, bottle haplomer-ligand complexes, and fusion proteins areadministered in parallel, ligands from different sets of haplomer-ligandcomplexes that have the same bio-orthogonal reactive group (or groupsthat do not react with each other, if different bio-orthogonalchemistries are employed for different sets of haplomer-ligandcomplexes) may be administered together, even at high concentrations,since they will not be reactive with each other. For example, if anazide-alkyne bio-orthogonal reactive system is employed for each set ofcorresponding haplomer-ligand complexes, all of the azide-containinghaplomer-ligand complexes may be formulated and administered together,and all of the alkyne-containing haplomer-ligand complexes may beformulated and administered together after sufficient dilution of theazides in the sample.

Production of effector proteins by the methods described herein canyield activities, such as, inducing an immune response, programmed celldeath, apoptosis, necrosis, lysis, growth inhibition, inhibition ofviral infection, inhibition of viral replication, inhibition of oncogeneexpression, modification of gene expression, inhibition of microbialinfection, and inhibition of microbe replication, as well ascombinations of these biological activities.

In some embodiments, the composition administered can include two ormore sets of corresponding haplomer-ligand complexes, bottlehaplomer-ligand complexes, and fusion proteins that target two or moretarget nucleic acid molecules. Two or more target nucleic acid moleculesmay be found within the same gene transcript, or alternatively ondistinct and separate transcripts. Two or more sets of correspondinghaplomer-ligand complexes, bottle haplomer-ligand complexes, and fusionproteins recognizing distinct nucleic acid target molecules within thesame cellular transcript may independently produce the same or differentproteins.

The abundance of target nucleic acid molecules may also limit the amountof active protein produced by templated assembly. In some embodiments,there is an average of at least 5 copies of target nucleic acidmolecules per target compartment. The dosage and concentration of thecomposition administered can take the availability of the target nucleicacid molecules into account.

In some embodiments, methods of delivering haplomer-ligand complexes,bottle haplomer-ligand complexes, and fusion proteins or a compositioncomprising one or more sets of the same to a pathogenic cell isdisclosed. The methods can include administering a therapeuticallyeffective amount of a set or multiple sets of correspondinghaplomer-ligand complexes, bottle haplomer-ligand complexes, and fusionproteins compositions to the pathogenic cell. In some embodiments, themethods can also include detecting the presence or absence of the targetnucleic acid molecule prior to administering the haplomer-ligandcomplexes, bottle haplomer-ligand complexes, and fusion proteinscomposition.

Pharmaceutical compositions may be administered by one of the followingroutes: oral, topical, systemic (e.g, transdermal, intranasal, or bysuppository), or parenteral (e.g. intramuscular, subcutaneous, orintravenous injection). Compositions may take the form of tablets,pills, capsules, semisolids, powders, sustained release formulations,solutions, suspensions, elixirs, aerosols, or any other appropriatecompositions; and comprise at least one compound in combination with atleast one pharmaceutically acceptable excipient. Suitable excipients arewell known to persons of ordinary skill in the art, and they, and themethods of formulating the compositions, may be found in such standardreferences as Remington: The Science and Practice of Pharmacy, A.Gennaro, ed., 20th edition, Lippincott, Williams & Wilkins,Philadelphia, Pa. Suitable liquid carriers, especially for injectablesolutions, include water, aqueous saline solution, aqueous dextrosesolution, and glycols.

Pharmaceutical compositions suitable for injection may include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases, the composition should be sterileand should be fluid to the extent that easy syringeability exists. Thecomposition should be stable under the conditions of manufacture andstorage and should be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, water, ethanol, polyol(for example, glycerol, propylene glycol, and liquid polyetheyleneglycol, and the like), suitable mixtures thereof, and vegetable oils.The proper fluidity can be maintained, for example, by the use of acoating such as lecithin, by the maintenance of the required particlesize in the case of dispersion and by the use of surfactants. Preventionof the action of microorganisms can be achieved by various antibacterialand antifungal agents. In many cases, isotonic agents can be included,for example, sugars, polyalcohols such as mannitol, sorbitol, sodiumchloride in the composition. Prolonged absorption of the injectablecompositions can be brought about by including in the composition anagent which delays absorption, for example, aluminum monostearate andgelatin.

Sterile injectable solutions can be prepared by incorporating thecomposition containing the haplomer-ligand complexes, bottlehaplomer-ligand complexes, and fusion proteins in a suitable amount inan appropriate solvent with one or a combination of ingredientsenumerated above. Generally, dispersions are prepared by incorporatingthe composition into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above

When the composition containing the haplomer-ligand complexes, bottlehaplomer-ligand complexes, and fusion proteins is suitably protected, asdescribed above, the composition can be formulated for oraladministration, for example, with an inert diluent or an assimilableedible carrier. The composition and other ingredients can also heenclosed in a hard or soft shell gelatin capsule, compressed intotablets, or incorporated directly into the subject's diet. For oraltherapeutic administration, the composition can be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.The percentage of the compositions and preparations can, of course, bevaried. The amount of haplomer-ligand complexes, bottle haplomer-ligandcomplexes, and fusion proteins in such therapeutically usefulcompositions is such that a suitable dosage will be obtained.

It may be advantageous to formulate compositions in dosage unit form forease of administration and uniformity of dosage. Each dosage unit formcontains a predetermined quantity of the haplomer-ligand complexes,bottle haplomer-ligand complexes, and fusion proteins calculated toproduce the amount of active effector product in association with apharmaceutical carrier. The specification for the novel dosage unitforms is dependent on the unique characteristics of the targetedtemplated assembly composition, and the particular therapeutic effect tobe achieved. Dosages are determined by reference to the usual dose andmanner of administration of the ingredients.

The haplomer-ligand complexes, bottle haplomer-ligand complexes, andfusion proteins compositions may comprise pharmaceutically acceptablecarriers, such that the carrier can be incorporated into the compositionand administered to a patient without causing unacceptable biologicaleffects or interacting in an unacceptable manner with other componentsof the composition. Such pharmaceutically acceptable carriers typicallyhave met the required standards of toxicological and manufacturingtesting, and include those materials identified as suitable inactiveingredients by the U.S. Food and Drug Administration.

The haplomer-ligand complexes, bottle haplomer-ligand complexes, andfusion proteins can also be prepared as pharmaceutically acceptablesalts. Such salts can be, for example, a salt prepared from a base or anacid which is acceptable for administration to a patient, such as amammal (e.g., salts having acceptable mammalian safety for a givendosage regime). However, it is understood that the salts covered hereinare not required to be pharmaceutically acceptable salts, such as saltsof the haplomers that are not intended for administration to a patient.Pharmaceutically acceptable salts can be derived from pharmaceuticallyacceptable inorganic or organic bases and from pharmaceuticallyacceptable inorganic or organic acids. In addition, when a haplomercontains both a basic moiety, such as an amine, and an acidic moietysuch as a carboxylic acid, zwitterions may be formed and are includedwithin the term “salt” as used herein. Salts derived frompharmaceutically acceptable inorganic bases can include ammonium,calcium, copper, ferric, ferrous, lithium, magnesium, manganic,manganous, potassium, sodium, and zinc salts, and the like. Saltsderived from pharmaceutically acceptable organic bases can include saltsof primary, secondary and tertiary amines, including substituted amines,cyclic amines, naturally-occurring amities and the like, such asarginine, betaine, caffeine, choline, N,N-dibenzylethylenediamine,diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol,ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine,glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperadine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like. Salts derived frompharmaceutically acceptable inorganic acids can include salts of boric,carbonic, hydrohalic (hydrobromic, hydrochloric, hydrofluoric orhydroiodic), nitric, phosphoric, sulfamic and sulfuric acids. Saltsderived from pharmaceutically acceptable organic acids can include saltsof aliphatic hydroxyl acids (e.g., citric, gluconic, glycolic, lactic,lactobionic, malic, and tartaric acids), aliphatic monocarboxylic acids(e.g., acetic, butyric, formic, propionic and trifluoroacetic acids),amino acids (e.g., aspartic and glutamic acids), aromatic carboxylicacids (e.g., benzoic, p-chlorobenzoic, diphenylacetic, gentisic,hippuric, and triphenylacetic acids), aromatic hydroxyl acids (e.g.,o-hydroxybenzoic, p-hydroxybenzoic, 1-hydroxynaphthalene-2-carboxylicand 3-hydroxynaphthalene-2-carboxylic acids), ascorbic, dicarboxylicacids (e.g., fumaric, maleic, oxalic and succinic acids), glucoronic,mandelic, mucic, nicotinic, orotic, pamoic, pantothenic, sulfonic acids(e.g., benzenesulfonic, camphorsulfonic, edisylic, ethanesulfonic,isethionic, methanesulfonic, naphthalenesuffonic,naphthalene-1,5-disulfonic, naphthalene-2,6-disulfonic andp-toluenesulfonic acids), xinafoic acid, and the like.

The effector proteins generated by the processes described herein (viadimerization or folding) is the trigger that drives a desired action.Some examples of desired protein activity can include, but are notlimited to, inducing an immune response, programmed cell death,apoptosis, non-specific or programmed necrosis, lysis, growthinhibition, inhibition of viral infection, inhibition of viralreplication, inhibition of oncogene expression, modification of geneexpression, inhibition of microbial infection, and inhibition of microbereplication, as well as combinations of these biological activities. Insome embodiments, the protein produced can serve as a ligand for anantibody to induce an immune response at the site of the pathogeniccells, or to localize antibody-directed therapies, such as an antibodybearing a therapeutic payload, to the site of the pathogenic cells. Insome embodiments, the protein produced can modulate expression of atarget gene. In some embodiments, the protein produced can regulateenzyme activity, gene/protein expression, molecular signaling, andmolecular interactions.

In some embodiments involving the use of the dimerization domains FKBPor FRB, the domains were altered in specific ways to improve theirperformances when expressed as fusion proteins with protein fragments ofinterest. In particular, it was observed that both FKBP and FRB possesssingle cysteine residues. Many of the protein fragments of interest,including but not limited to Gaussia luciferase and heavy and lightchains of immunoglobulins, themselves possess multiple cysteines whichin some cases are structurally important for the formation of disulfidebonds and folding. Since only single cysteines were present in both FKBPand FRB, it could not the case that disulfide bonds were essential forthe folding of the dimerization domains themselves. Accordingly, it wasconsidered possible that the FKBP and FRB cysteines could potentiallyinterfere with folding maturation of desired fusion proteins byformation of structurally incorrect internal cross-disulfide bonds. Inturn, conservative replacement of the FKBP and FRB cysteines withalternative amino acid residues could be beneficial, provided thechanges were compatible with the stabilities and ligand-bindingactivities of these domains. Therefore, in some embodiments, mutants ofFKBP, in addition to the F36V mutation, also contained a cysteine toserine mutation (C225). In certain other embodiments, a cysteine toalanine mutation (C22A) or a cysteine to valine mutation (C22V) may alsobe used. In an analogous manner, in some embodiments mutants of FRB areused with a cysteine to serine mutation (C61S), or in In certain otherembodiments, a cysteine to alanine mutation (C61A) or a cysteine tovaline mutation (C61V).

In all cases, mutagenesis was effected in a standard fashion byamplifications of two overlapping blocks of coding sequences where oneof the primers specified the desired coding sequence changes. Asubsequent round of PCR enabled precise fusion of the sequence blocks,confirmed by sequencing.

The following representative embodiments are presented:

Embodiment 1. A haplomer-ligand complex comprising: a haplomer, whereinthe haplomer comprises a polynucleotide that is substantiallycomplementary to a target nucleic acid molecule; and a ligand linked tothe 5′ or 3′ terminus of the haplomer, wherein the ligand comprises aligand partner binding site.

Embodiment 2. The haptomer-ligand complex of embodiment 1 wherein thepolynucleotide comprises DNA nucleotides, RNA nucleotides,phosphorothioate-modified nucleotides, 2-O-alkylated RNA nucleotides,halogenated nucleotides, locked nucleic acid nucleotides (LNA), peptidenucleic acids (PNA), morpholino nucleic acid analogues (morpholines),pseudouridine nucleotides, xanthine nucleotides, hypoxanthinenucleotides, 2-deoxyinosine nucleotides, DNA analogs with L-ribose(L-DNA), Xeno nucleic acid (XNA) analogues, or other nucleic acidanalogues capable of base-pair formation, or artificial nucleic acidanalogues with altered backbones, or any combination thereof.

Embodiment 3. The haptomer-ligand complex of embodiment 1 or embodiment2 wherein the ligand is a small molecule ligand or an interactiveprotein domain.

Emobdiment 4. The haplomer-ligand complex of embodiment 3 wherein thesmall molecule ligand is less than about 2500 Daltons.

Embodiment. 5. The haplomer-ligand complex of emobodiment 4 wherein thesmall molecule ligand is a small molecule, a peptide having less thanabout 20 amino acid residues, a naturally- or artificially-modifiedpeptide, a peptidomimetic, a glycan, an organic enzyme cofactor, or anartificially-derived small molecular ligand.

Embodiment 6. The haplomer-ligand complex of embodiment 3 wherein thesmall molecule ligand is an FKM monovalent ligand.

Embodiment 7. The haplomer-ligand complex of embodiment 6wherein the FKMmonovalent ligand is FKM-NHS, FKM-sulfo-NHS, FKM-PEG3-NHS, or MFL2,wherein:

FKM-NHS is

where m is from 3 to

6; FKM-sulfa-NHS is

where m is from 3 to 6; FKM-PEG3-NHS is

where n is from 1 to 6; and MFL2 is

Embodiment 8. The haplomer-ligand complex of any one of embodiments 1 to7 wherein the ligand further comprises a bio-orthogonal moiety.

Embodiment 9. The haplomer-ligand complex of embodiment 8 wherein thebio-orthogonal moiety is an azide, an alkyne, a cyclooctyne, a nitron, anorbornene, an oxanorbornadiene, a phosphine, a dialkyl phosphine, atrialkyl phosphine, a phosphinothiol, a phosphinophenol, a cyclooctene,a nitrile oxide, a thioester, a tetrazine, isonitrile, a tetrazole, or aquadricyclane, or any derivative thereof.

Embodiment 10. The haplomer-ligand complex of embodiment 8 wherein theligand is an FKM monovalent ligand chosen from FKM-PEG3-MTZ-NHS andFKM-PEG3-TCO-NHS, wherein: FKM-PEG3-MTZ-NHS is

where x is from 1 to 6; and FKM-PEG3-TCO-NHS is

where x is from 1 to 6.

Embodiment 11. The haplomer-ligand complex of embodiment 3 wherein theinteractive protein domain comprises less than 100 amino acid residues.

Embodiment 12. The haplomer-ligand complex of embodiment 11 wherein theinteractive protein domain is a leucine zipper domain.

Embodiment 13. The haplomer-ligand complex of embodiment 12 wherein theinteractive protein domain is a c-jun domain, a c-fos domain, a c-mycdomain, a c-max domain, an NZ domain, or a CZ domain.

Embodiment 14. The haplomer-ligand complex of embodiment 13 wherein theNZ domain comprises the amino acid sequenceALKKELQANKKELAQLKWELQALKKELAQ (SEQ ID NO:10), and the CZ domaincomprises the amino acid sequence EQLEKKLQALEKK LAQLEWKNQALEKKLAQ (SEQID NO:11).

Embodiment 15. The haplomer-ligand complex of embodiment 12 wherein theN-terminus of the c-jun domain is linked to the 5′ or 3′ terminus of thepolynucleotide of the haplomer.

Embodiment 16. The haplomer-ligand complex of embodiment 15 wherein thec-jun domain comprises the amino acid sequenceCSGGASLERIARLEEKVKTLKAQNSELAST ANMLREQVAQLKQKGAP (SEQ ID NO:1),CSGGASLERIARLEEKVKSEKAQNSENAST ANMLREQVAQLKQKGAP (SEQ ID NO;4), orCSGASLERIARLEEKVKSFKAQNSENAST ANMLREQVAQLKQKGAP (SEQ ID NO:12).

Embodiment 17. The haplomer-ligand complex of embodiment 12 wherein theC-terminus of the c-jun domain is linked to the 5′ or 3′ terminus of thepolynucleotide of the haplomer.

Embodiment 18, The haplomer-ligand complex of embodiment 17 wherein thec-jun domain comprises the amino acid sequenceSGASLERIARLEEKVKTLKAQNSELASTANM LREQVAQLKQKQAPSGGC (SEQ ID NO:2) orSGASLERIARLEEKVKSFKAQNSENASTA NMLREQVAQLKQKGAPSGGC (SEQ ID NO:5).

Embodiment 19. A composition or kit comprising a first haplomer-ligandcomplex of embodiment 1 and a second haplomer-ligand complex ofembodiment 1, wherein: the ligand of the first haplomer-ligand complexis linked to the 5′ terminus of the polynucleotide of the firsthaplomer-ligand complex; and the ligand of the second haplomer-ligandcomplex is linked to the 3′ terminus of the polynucleotide of the secondhaplomer-ligand complex.

Embodiment 20. The composition or kit of embodiment 19, wherein thepolynucleotide of the first haplomer-ligand complex is substantiallycomplementary to the polynucleotide of the second haplomer-ligandcomplex.

Embodiment 21. The composition or kit of embodiment 19, wherein thepolynucleotide of the first haplomer-ligand complex is substantiallycomplementary to a target nucleic acid molecule, and the polynucleotideof the second haplomer-ligand complex is substantially complementary tothe target nucleic acid molecule at a site in spatial proximity to thepolynucleotide of the first haplomer-ligand complex.

Embodiment 22. The composition or kit of any one of embodiments 19 to21, wherein the, polynucleotides of the first haplomer-ligand complexand second haplomer-ligand complex comprise DNA nucleotides, RNAnucleotides, phosphorothioate-modified nucleotides, 2-O-alkylated RNAnucleotides, halogenated nucleotides, locked nucleic acid nucleotides(LNA), peptide nucleic acids (PNA), morpholine nucleic acid analogues(morpholines), pseudouridine nucleotides, xanthine nucleotides,hypoxanthine nucleotides, 2-deoxyinosine nucleotides, DNA analogs withL-ribose (L-DNA), Xeno nucleic acid (XNA) analogues, or other nucleicacid analogues capable of base-pair formation, or artificial nucleicacid analogues with altered backbones, or any combination thereof.

Embodiment 23. The composition or kit of any one of embodiments 19 to22, wherein both ligands are small molecule ligands or both ligands areinteractive protein domains.

Embodiment 24. The composition or kit of embodiment 23 wherein bothsmall molecule ligands are less than about 2500 Daltons.

Embodiment 25. The composition or kit of embodiment 24 wherein bothsmall molecule ligands are small molecules, peptides having less thanabout 20 amino acid residues, naturally- or artificially-modifiedpeptides, peptidomimetics, glycans, organic enzyme cofactors, orartificially-derived small molecular ligands.

Embodiment 26. The composition or kit of embodiment 23 wherein bothsmall molecule ligands are FKM monovalent ligands.

Embodiment 27. The composition or kit of embodiment 26 wherein each FKMmonovalent ligand is, independently, FKM-NHS, FKM-sulfo-NHS,FKM-PEG3-NHS, or MFL2, wherein: FKM-NHS is

where in is from 3 to 6; FKM-sulfo-NHS is

where m is from 3 to 6; FKM-PEG-3-NHS is

where n is from 1 to 6; and MFL2 is

Embodiment 28. The composition or kit of any one of embodiments 19 to 27wherein: the ligand of the first haplomer-ligand complex furthercomprises a bio-orthogonal moiety; and the ligand of the secondhaplomer-ligand complex further comprises a bio-orthogonal moiety;wherein the bio-orthogonal moiety of the first haplomer-ligand complexis reactable with the bio-orthogonal moiety of the secondhaplomer-ligand complex.

Embodiment 29. The composition or kit of embodiment 28 wherein thereadable bio-orthogonal moietys are chosen from an azide, an alkyne, acyclooctyne, a nitrone, a norbornene, an oxanorbornadiene, a phosphine,a dialkyl phosphine, a trialkyl phosphine, a phosphinothiol, aphosphinophenol, a cyclooctene, a nitrile oxide, a thioester, atetrazine, an isonitrile, a tetrazole, or a quadricyciane, or anyderivative thereof.

Embodiment 30. The composition or kit of embodiment 28 wherein theligand of one of the first haplomer-ligand complex or secondhaplomer-ligand complex is an FKM monovalent ligand that isFKM-PEG3-MTZ-NHS and the ligand of the other of the firsthaplomer-ligand complex or second haplomer-ligand complex is an FKMmonovalent ligand that is FKM-PEG3-TCO-NHS, wherein: FKM-PEG3-MTZ-NHS is

where x is from 1 to 6; and FKM-PEG3-TCO-NHS is

where x is from 1 to 6.

Embodiment 31. The composition or kit of embodiment 23 wherein bothinteractive protein domains each comprise less than 100 amino acidresidues.

Embodiment 32. The composition or kit of embodiment 31 wherein bothinteractive protein domains are leucine zipper domains.

Embodiment 33. The composition or kit of embodiment 32 wherein eachinteractive protein domain is, independently, a c-jun domain, a c-fosdomain, a c-myc domain, a c-max domain, an NZ domain, or a CZ domain.

Embodiment 34. The composition or kit of embodiment 33 wherein theN-terminus of the interactive protein domain of the firsthaplomer-ligand complex is linked to the 5′ terminus of thepolynucleotide of the first haplomer-ligand complex, and the N-terminusof the interactive protein domain of the second haplomer-ligand complexis linked to the 3′ terminus of the polynucleotide of the secondhaplomer-ligand complex.

Embodiment 35. The composition or kit of embodiment 33 wherein theC-terminus of the interactive protein domain of the firsthaplomer-ligand complex is linked to the 5′ terminus of thepolynucleotide of the first haplomer-ligand complex, and the C-terminusof the interactive protein domain of the second haplomer-ligand complexis linked to the 3′ terminus of the polynucleotide of the secondhaplomer-ligand complex.

Embodiment 36. The composition or kit of embodiment 33 wherein: theC-terminus of the interactive protein domain of the firsthaplomer-ligand complex is linked to the 5′ terminus of thepolynucleotide of the first haplomer-ligand complex, and the N-terminusof the interactive protein domain of the second haplomer-ligand complexis linked to the 3′ terminus of the polynucleotide of the secondhaplomer-ligand complex; or the N-terminus of the interactive proteindomain of the first haplomer-ligand complex is linked to the 5′ terminusof the polynucleotide of the first haplomer-ligand complex, and theC-terminus of the interactive protein domain of the secondhaplomer-ligand complex is linked to the 3′ terminus of thepolynucleotide of the second haplomer-ligand complex.

Embodiment 37. The composition or kit of any one of embodiments 33 to 36wherein the NZ domain comprises the amino acid sequenceALKKELQANKKELAQLKWELQALKKE LAQ (SEQ ID NO:10), and the CZ domaincomprises the amino acid sequence EQLEKKLQAL EKKLAQLEWKNQALEKKLAQ (SEQID NO:11).

Embodiment 38. The composition or kit of any one of embodiments 33 to 36wherein the ligand linked to the polynucleotide of the firsthaplomer-ligand complex is a c-jun domain, and the ligand linked to thepolynucleotide of the second haplomer-ligand complex is a c-jun domain.

Embodiment 39. The composition or kit of any one of embodiments 33 to 36wherein the c-jun domain comprises the amino acid sequenceCSGGASLERIARLEEKVKTLKAQNS ELASTANMLREQVAQLKQKGAP (SEQ ID NO:1),CSGGASLERIARLEEKVKSFKAQNS ENASTANMLREQVAQLKQKGAP (SEQ ID NO:4), orASLERIARLEEKVKSFKAQNSEN ASTANMLREQVAQLKQKGAP (SEQ ID NO:16).

Embodiment 40. The composition or kit of embodiment 35 or embodiment 36wherein the C-terminus of the c-jun domain is linked to the 5′ or 3′terminus of the polynucleotides of either or both of the firsthaplomer-ligand complex and second haplomer-ligand complex.

Embodiment 41. The composition or kit of embodiment 40 wherein the c-jundomain comprises the amino acid sequenceSGASLERIARLEEKVKTLKAQNSELASTANMLREQ VAQLKQKGAPSGGC (SEQ ID NO:2) orGASLERIARLEEKVKSFKAQNSENASTANML REQVAQLKQKGAPSGGC (SEQ ID NO:5).

Embodiment 42. The composition or kit of any one of embodiments 33 to 36wherein the ligand linked to the polynucleotide of the firsthaplomer-ligand complex is a c-jun domain or a c-myc domain, and theligand linked to the polynucleotide of the second haplomer-ligandcomplex is a c-jun domain or a c-myc domain.

Embodiment 43. The composition or kit of embodiment 42 wherein theligand linked to the polynucleotide of the first haplomer-ligand complexor second haplomer-ligand complex is a c-jun domain, and the ligandlinked to the polynucleotide of the other of the first haplomer-ligandcomplex and second haplomer-ligand complex is a c-myc domain.

Embodiment 44. A bottle haplomer-ligand complex comprising: a) a bottlehaplomer, wherein the bottle haplomer comprises a polynucleotide,wherein the polynucleotide comprises: i) a first stem portion comprisingfrom about 10 to about 20 nucleotide bases; ii) an anti-target loopportion comprising from about 16 to about 40 nucleotide bases and havinga first end to which the first stem portion is linked, wherein theanti-target loop portion is substantially complementary to a targetnucleic acid molecule; and iii) a second stem portion comprising fromabout 10 to about 20 nucleotide bases linked to a second end of theanti-target loop portion, wherein the first stem portion issubstantially complementary to the second stem portion; and b) a ligandlinked to the terminal end of either the first stem portion or thesecond stem portion, wherein the ligand comprises a ligand partnerbinding wherein the T_(m) of the anti-target loop portion:target nucleicacid molecule is greater than the T_(m) of the first stem portion:secondstem portion.

Embodiment 45. The bottle haplomer-ligand complex of embodiment 44wherein the T_(m) of the first stem portion:second stem portionsubtracted from the T_(m) of the anti-target loop portion:target nucleicacid molecule is from about 10° C. to about 40° C.

Embodiment 46. The bottle haplomer-ligand complex of embodiment 44 orembodiment 45 wherein the T_(m) of the first stem portion:second stemportion is from about 40° C. to about 50° C.

Embodiment 47. The bottle haplomer-ligand complex of any one ofembodiments 44 to 46 wherein the T_(m) of the anti-target loopportion:target nucleic acid molecule is from about 60° C. to about 80°C.

Embodiment 48. The bottle haplomer-ligand complex of any one ofembodiments 44 to 47 wherein the T_(m) of the first stem portion:secondstem portion subtract from the T_(m) of the anti-target loopportion:target nucleic acid molecule is from about 10° C. to about 20°C.

Embodiment 49. The bottle haplomer-ligand complex of any one ofembodiments 44 to 48 wherein the first stem portion comprises from about12 to about 18 nucleotide bases.

Embodiment 50. The bottle haplomer-ligand complex of any one ofembodiments 44 to 49 wherein the anti-target loop portion comprises fromabout 18 to about 35 nucleotide bases.

Embodiment 51. The bottle haplomer-ligand complex of any one ofembodiments 44 to 50 wherein the second stem portion comprises fromabout 12 to about 18 nucleotide bases.

Embodiment 52. The bottle haplomer-ligand complex of any one ofembodiments 44 to 51 wherein the nucleotide bases of any one or more ofthe first stem portion, anti-target loop portion, and second stemportion are selected from the group consisting of DNA nucleotides, RNAnucleotides, phosphorothioate-modified nucleotides, 2-O-alkylated RNAnucleotides, halogenated nucleotides, locked nucleic acid nucleotides(LNA), peptide nucleic acids (PNA), morpholino nucleic acid analogues(morpholinos), pseudouridine nucleotides, xanthine nucleotides,hypoxanthine nucleotides, 2-deoxyinosine nucleotides, DNA analogs withL-ribose (L-DNA), Xeno nucleic acid (XNA) analogues, or other nucleicacid analogues capable of base-pair formation, or artificial nucleicacid analogues with altered backbones, or any combination. thereof.

Embodiment 53. The bottle haplomer-ligand complex of any one ofembodiments 44 to 52 further comprising a linker between any one or moreof the first stem portion and the anti-target loop portion, between theanti-target loop portion and the second stem portion, and between thesecond stem portion and the reactive effector moiety.

Embodiment 54. The bottle haplomer-ligand complex of embodiment 53wherein the linker is selected from the group consisting of an alkylgroup, an alkenyl group, an amide, an ester, a thioester, a ketone, anether, a thioether, a disulfide, an ethylene glycol, a cycloalkyl group,a benzyl group, a heterocyclic group, a maleimidyl group, a hydrazone, aurethane, azoles, an imine, a haloalkyl, and a carbamate, or anycombination thereof.

Embodiment 55. The bottle haplomer-ligand complex of any one ofembodiments 44 to 54 wherein the anti-target loop portion furthercomprises an internal hinge region, wherein the hinge region comprisesone or more nucleotides that are not complementary to the target nucleicacid molecule.

Embodiment 56. The bottle haplomer-ligand complex of embodiment 55wherein the hinge region comprises from about 1 nucleotide to about 6nucleotides.

Embodiment 57. The bottle haplomer-ligand complex of any one ofembodiments 44 to 56 which comprises the nucleotide sequence5′-ACTCGAGACGTCTCCTTGTCTTTGCTTTT CTTCAGGACACAGTGGCGAGACGTCTCGAGT-3′ (SEQID NO:6) or 5′-ACTCGAGACG TCTCCTTCCTGCCCCTCCTCCTGCTCCGAGACGTCTCGAGT-3′(SEQ ID NO:7).

Embodiment 58. The bottle haplomer-ligand complex of any one ofembodiments 44 to 57 wherein the ligand is a small molecule ligand or aninteractive protein domain.

Embodiment 59. The bottle haplomer-ligand complex of embodiment 58wherein the small molecule ligand is less than about 2500 Daltons.

Embodiment 60. The bottle haplomer-ligand complex of embodiment 59wherein the small molecule ligand is a small molecule, a peptide havingless than about 20 amino acid residues, a naturally- orartificially-modified peptide, a peptidomimetic, a glycan, an organicenzyme cofactor, or an artificially-derived small molecular ligand.

Embodiment 61. The bottle haplomer-ligand complex of embodiment 58wherein the small molecule ligand is an FKM monovalent ligand.

Embodiment 62. The bottle haplomer-ligand complex of embodiment 61wherein the FKM monovalent ligand is FKM-NHS, FKM-sulfo-NHS,FKM-PEG3-NHS, MFL2, wherein:

FKM-NHS is

where m is from 3 to 6; FKM-sulfa-NHS is

where m is from 3 to 6; FKM-PEG3-NHS is

where n is from 1 to 6; and MFL2. is

Embodiment 63. The bottle haplomer-ligand complex of any one ofembodiments 44 to 62 wherein the ligand further comprises abio-orthogonal moiety.

Embodiment 64. The bottle haplomer-ligand complex of embodiment 63wherein the bio-orthogonal moiety is an azide, an alkyne, a cyclooctyne,a nitrone, a norbornene, an oxanorbornadiene, a phosphine, a dialkylphosphine, a trialkyl phosphine, a phosphinothiol, a phosphinophenol, acyclooctene, a nitrile oxide, a thioester, a tetrazine, isonitrile, atetrazole, or a quadricyclane, or any derivative thereof.

Embodiment 65. The bottle haplomer-ligand complex of embodiment. 63wherein the ligand is an FKM monovalent ligand chosen fromFKM-PEG3-MTZ-NHS and FKM-PEG3-TCO-NHS, wherein: FKM-PEG-3-MTZ-NHS is

where x is front 1 to 6; and FKM-PEG3-TCO-NHS is

where x is from 1 to 6.

Embodiment 66. The bottle haplomer-ligand complex of embodiment 58wherein the interactive protein domain comprises less than 100 aminoacid residues.

Embodiment 67. The bottle haplomer-ligand complex of embodiment 66wherein the interactive protein domain is a leucine zipper domain.

Embodiment 68. The bottle haplomer-ligand complex of embodiment 67wherein the interactive protein domain is a c-jun domain, a c-fosdomain, a c-myc domain, a c-max domain, an NZ domain, or a CZ domain.

Embodiment 69. The bottle haplomer-ligand complex of embodiment 68wherein the NZ domain comprises the amino acid sequenceALKKELQANKKELAQLKWELQALKKEL AQ (SEQ ID NO:10), and the CZ domaincomprises the amino acid sequence EQLEKKLQALE KKLAQLEWKNQALEKKLAQ (SEQID NO:11).

Embodiment 70. The bottle haplomer-ligand complex of embodiment 67wherein the N-terminus of the c-jun domain is linked to the 5′ or 3′terminus of the polynucleotide of the bottle haplomer.

Embodiment 71. The bottle haplomer-ligand complex of embodiment 70wherein the c-jun domain comprises the amino acid sequenceCSGGASLERIARLEEKVKTLKAQNSELAST ANMLREQVAQLKQKGAP (SEQ ID NO:1),CSGGASLERIARLEEKVKSFKAQNSEN ASTANMLREQVAQLKQKGAP (SEQ ID NO:4), orCSGASLERIARLEEKVKSFKAQNSE NASTANMLREQVAQLKQKGAP (SEQ ID NO:12).

Embodiment 72. The bottle haplomer-ligand complex of embodiment 67wherein the C-terminus of the c-jun domain is linked to the 5′ or 3′terminus of the polynucleotide of the bottle haplomer.

Embodiment 73. The bottle haplomer-ligand complex of embodiment 72wherein the c-jun domain comprises the amino acid sequenceSGASLERIARLEEKVKTLKAQNSELASTAN MLREQVAQLKQKGAPSGGC (SEQ ID NO:2) orSGASLERIARLEEKVKSFKAQNSENAST ANMLREQVAQLKQKGAPSGGC (SEQ ID NO:5).

Embodiment 74. A composition or kit comprising: a bottle haplomer-ligandcomplex of any one of embodiments 44 to 73; and a second haplomer-ligandcomplex comprising: a nucleotide portion comprising from about 6 toabout 20 nucleotide bases that is substantially complementary to thestem portion of the bottle haplomer-ligand complex that is linked to theligand of the bottle haplomer-ligand complex; and a ligand linked to the5′ or 3′ terminus of the nucleotide portion of the secondhaplomer-ligand complex, wherein the ligand comprises a ligand partnerbinding site; wherein the T_(m) of the second haplomer-ligandcomplex:first or second stem portion linked to the ligand of the bottlehaplomer-ligand complex is less than or equal to the T_(m) of the firststem portion:second stem portion of the bottle haplomer-ligand complex.

Embodiment 75. The composition or kit of embodiment 74 wherein the T_(m)of the duplex formed by the second haplomer-ligand complex and the firstor second stem portion of the bottle haplomer-ligand complex linked tothe ligand subtracted from the T_(m) of the first stem portion:secondstem portion of the bottle haplomer-ligand complex is from about 0° C.to about 20° C.

Embodiment 76. The composition or kit of embodiment 74 or embodiment 75wherein the T_(m) of the duplex formed by the second haplomer-ligandcomplex and the first or second stem portion of the bottlehaplomer-ligand complex linked to the ligand is from about 30° C. toabout 20 40° C.

Embodiment 77. The composition or kit of any one of embodiments 74 to 76wherein the T_(m) of the duplex formed by the second haplomer-ligandcomplex and the first or second stem portion of the bottlehaplomer-ligand complex linked to the ligand subtracted from the T_(m)of the first stem portion:second stem portion of the bottlehaplomer-ligand complex is from about 5° C. to about 10° C.

Embodiment 78. The composition or kit of any one of embodiments 74 to 77wherein the polynucleotide of the second haplomer-ligand complexcomprises from about 8 to about 15 nucleotide bases.

Embodiment 79. The composition or kit of any one of embodiments 74 to 77wherein the polynucleotides of the bottle haplomer-ligand complex andthe second haplomer-ligand complex comprise DNA nucleotides, RNAnucleotides, phosphorothioate-modified nucleotides, 2-O-alkylated RNAnucleotides, halogenated nucleotides, locked nucleic acid nucleotides(LNA), peptide nucleic acids (PNA), morpholino nucleic acid analogues(morpholinos), pseudouridine nucleotides, xanthine nucleotides,hypoxanthine nucleotides, 2-deoxyinosine nucleotides, DNA analogs withL-ribose (L-DNA), Xeno nucleic acid (XNA) analogues, or other nucleicacid analogues capable of base-pair formation, or artificial nucleicacid analogues with altered backbones, or any combination thereof.

Embodiment 80. The composition or kit of any one of embodiments 74 to79, wherein both ligands are small molecule ligands or both ligands areinteractive protein domains.

Embodiment 81. The composition or kit of embodiment 80 wherein bothsmall molecule ligands are less than about 2500 Daltons.

Embodiment 82. The composition or kit of embodiment 81 wherein bothsmall molecule ligands are small molecules, peptides having less thanabout 20 amino acid residues, naturally- or artificially-modifiedpeptides, peptidomimetics, glycans, organic enzyme cofactors, orartificially-derived small molecular ligands.

Embodiment 83. The composition or kit of embodiment 80 wherein bothsmall molecule ligands are FKM monovalent ligands.

Embodiment 84. The composition or kit of embodiment 83 wherein each FKMmonovalent ligand is, independently, FKM-NHS, FKM-sulfo-NHS,FKM-PEG3-NHS, or MFL2, wherein: FKM-NHS is

where m is from 3 to 6; FKM-sulfo-NHS is

where m is from 3 to 6;

FKM-PEG3-NHS is

where n is from l to 6; and MFL2 is

Embodiment 85. The composition or kit of any one of embodiments 74 to 84wherein: the ligand of the bottle haplomer-ligand complex furthercomprises a bio-orthogonal moiety; and the ligand of the secondhaplomer-ligand complex further comprises a bio-orthogonal moiety;wherein the bio-orthogonal moiety of the bottle haplomer-ligand complexis reactable with the bio-orthogonal moiety of the secondhaplomer-ligand complex.

Embodiment 86. The composition or kit of embodiment 85 wherein thereactable bio-orthogonal moietys are chosen from an azide, an alkyne, acyclooctyne, a nitrone, a norbornene, an oxanorbornadiene, a phosphine,a dialkyl phosphine, a trialkyl phosphine, a phosphinothiol, aphosphinophenol, a cyclooctene, a nitrile oxide, a thioester, atetrazine, an isonitrile, a tetrazole, or a quadricyclane, or anyderivative thereof.

Embodiment 87. The composition or kit of embodiment 85 wherein theligand of one of the bottle haplomer-ligand complex and secondhaplomer-ligand complex is an FKM monovalent ligand that isFKM-PEG3-MTZ-NHS and the ligand of the other of the bottlehaplomer-ligand complex and second haplomer-ligand complex is an FKMmonovalent ligand that is FKM-PEG3-TCO-NHS, wherein: FKM-PEG3-MTZ-NHS is

where x is from 1 to 6; and FKM-PEG3-TCO-NHS is

where x is from 1. to 6.

Embodiment 88. The composition or kit of embodiment 80 wherein bothinteractive protein domains each comprise less than 100 amino acidresidues.

Embodiment 89. The composition or kit of embodiment 88 wherein bothinteractive protein domains are leucine zipper domains.

Embodiment 90. The composition or kit of embodiment 89 wherein eachinteractive protein domain is, independently, a c-jun domain, a c-fosdomain, a c-myc domain, a c-max domain, an NZ domain, or a CZ domain.

Embodiment 91. The composition or kit of embodiment 90 wherein theN-terminus of the interactive protein domain of the bottlehaplomer-ligand complex is linked to the 5′ terminus of thepolynucleotide of the bottle haplomer-ligand complex, and the N-terminusof the interactive protein domain of the second haplomer-ligand complexis linked to the 3′ terminus of the polynucleotide of the secondhaplomer-ligand complex.

Embodiment 92. The composition or kit of embodiment 90 wherein theC-terminus of the interactive protein domain of the bottlehaplomer-ligand complex is linked to the 5′ terminus of thepolynucleotide of the bottle haplomer-ligand complex, and the C-terminusof the interactive protein domain of the second haplomer-ligand complexis linked to the 3′ terminus of the polynucleotide of the secondhaplomer-ligand complex.

Embodiment 93. The composition or kit of embodiment 90 wherein: theC-terminus of the interactive protein domain of the bottlehaplomer-ligand complex is linked to the 5′ terminus of thepolynucleotide of the bottle haplomer-ligand complex, and the N-terminusof the interactive protein domain of the second haplomer-ligand complexis linked to the 3′ terminus of the polynucleotide of the secondhaplomer-ligand complex; or the N-terminus of the interactive proteindomain of the bottle haplomer-ligand complex is linked to the 5′terminus of the polynucleotide of the bottle haplomer-ligand complex,and he C-terminus of the interactive protein domain of the secondhaplomer-ligand complex is linked to the 3′ terminus of thepolynucleotide of the second haplomer-ligand complex.

Embodiment 94. The composition or kit of any one of embodiments 90 to 93wherein the NZ domain comprises the amino acid sequenceALKKELQANKKELAQLKWELQALKK ELAQ (SEQ ID NO:10), and the CZ domaincomprises the amino acid, sequence EQLEKKLQALEKKLAQLEWKNQALEKKLAQ (SEQID NO:11).

Embodiment 95. The composition or kit of any one of embodiments 90 to 93wherein the ligand linked to the polynucleotide of the bottlehaplomer-ligand complex is a c-jun domain, and the ligand linked to thepolynucleotide of the second haplomer-ligand complex is a c-jun domain.

Embodiment 96. The composition or kit of any one of embodiments 90 to 93wherein the c-jun domain comprises the amino acid sequenceCSGGASLERIARLEEKVKTLKAQNSE LASTANMLREQVAQLKQKGAP (SEQ ID NO:1),CSGGASLERIARLEEKVKSFKAQNSE NASTANMLREQVAQLKQKGAP (SEQ ID NO:4), orASLERIARLEEKVKSFKAQNSENAS TANMLREQVAQLKQKGAP (SEQ ID NO:12).

Embodiment 97. The composition or kit of embodiment 92 or embodiment 93wherein the C-terminus of the c-jun domain is linked to the 5′ or 3′terminus of the polynucleotides of either or both of the bottlehaplomer-ligand complex and second haplomer-ligand complex.

Embodiment 98. The composition or kit of embodiment 97 wherein the c-jundomain comprises the amino acid sequenceSGASLERIARLEEKVKTLKAQNSELASTANMLREQV AQLKQKGAPSGGC (SEQ ID NO:2) orSGASLERIARLEEKVKSFKAQNSENASTANML REQVAQLKQKGAPSGGC (SEQ ID NO:5).

Embodiment 99. The composition or kit of any one of embodiments 90 to 93wherein the ligand linked to the polynucleotide of the bottlehaplomer-ligand complex is a c-jun domain or a c-myc domain, and theligand linked to the polynucleotide of the second haplomer-ligandcomplex is a c-jun domain or a c-myc domain.

Embodiment 100. The composition or kit of embodiment 99 wherein theligand linked to the polynucleotide of one of the bottle haplomer-ligandcomplex and second haplomer-ligand complex is a c-jun domain, and theligand linked to the polynucleotide of the other of the bottlehaplomer-ligand complex and second haplomer-ligand complex is a c-mycdomain.

Embodiment 101. The kit of any one of embodiments 74 to 100 wherein: thepolynucleotide of the bottle haplomer-ligand complex comprises thenucleotide sequence of5′-ACTCGAGACGTCTCCTTGTCTTTGCTTTTCTTCAGGACACAGTGGCGAGACGT CTCGAGT-3′ (SEQID NO:6), and the polynucleotide of the second haplomer-ligand complexcomprises the nucleotide sequence of 5′-AGCTCTCGAGT-3′ (SEQ ID NO:8); orthe polynucleotide of the bottle haplomer-ligand complex comprises thenucleotide sequence of5′-ACTCGAGACGTCTCCTTCCTGCCCCTCCTCCTGCTCCGAGACGTCTCGAGT-3′ (SEQ ID NO:7),and the polynucleotide of the second haplomer-ligand complex comprisesthe nucleotide sequence 5′-GACGTCTCGAGT-3′ (SEQ ID NO:9).

Embodiment 102. A compound having the formula:

where m is from 3 to 6.

Embodiment 103. A compound having the formula:

where m is from 3 to 6.

Embodiment 104. A compound having the formula:

where n is from 1 to 6.

Embodiment 105. A compound having the formula:

where x is from 1 to 6.

Embodiment 106, A compound having the formula:

where x is from 1 to 6.

Embodiment 106B. A compound having the formula:

Embodiment 107. A fusion protein comprising a fragment of a protein ofinterest fused to a ligand binding domain, wherein: the ligand bindingdomain is a ligand binding domain for small molecule ligands; or theligand binding domain is an interactive protein domain.

Embodiment 108. The fusion protein of embodiment 107 wherein the ligandbinding domain is a ligand binding domain for small molecule ligands.

Embodiment 109. The fusion protein of embodiment 108 wherein the ligandbinding domain is an FKBP domain or an FRB domain.

Embodiment 110. The fusion protein of embodiment 109 wherein the FKBPdomain is a mutant FKBP domain.

Embodiment 110B. The fusion protein of embodiment 108 wherein the FKBPdomain comprises a C22S, C22A, or C22V substitution, or wherein the FRBdomain comprises a C61S, C61A, or C61V substitution.

Embodiment 111. The fusion protein of embodiment 110 wherein the mutantFKBP domain is the F36\1 FKBP mutant domain comprising the amino acidsequence GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO:14) or MGVQVETISPGDGR.TFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO:15).

Embodiment 112. The fusion protein of embodiment 107 wherein the ligandbinding domain is an interactive protein domain.

Embodiment 113. The fusion protein of embodiment 112 wherein theinteractive protein domain comprises less than 100 amino acid residues.

Embodiment 114. The fusion protein of embodiment 112 wherein theinteractive protein domain is a leucine zipper domain.

Embodiment 115. The fusion protein of embodiment 114 wherein theinteractive protein domain is a c-jun domain, a c-fos domain, a c-mycdomain, a c-max domain, an NZ domain, or a CZ domain.

Embodiment 116. The fusion protein of embodiment 115 wherein theinteractive protein domain is fused to the N-terminus of the protein ofinterest.

Embodiment 117. The fusion protein of embodiment 115 wherein theinteractive protein domain is fused to the C-terminus of the protein ofinterest.

Embodiment 118. The fusion protein of any one of embodiments 115 to 117wherein the NZ domain comprises the amino acid sequenceALKKELQANKKELAQLKWELQALKKE LAQ (SEQ ID NO:10), and the CZ domaincomprises the amino acid sequence EQLEKKLQAL EKKLAQLEWKNQALEKKLAQ (SEQID NO:11).

Embodiment 119. The fusion protein of any one of embodiments 115 to 117wherein the c-jun domain comprises the amino acid sequenceCSGGASLERIARLEEKVKTLKAQNSEL ASTANMLREQVAQLKQKGAP (SEQ ID NO:1),CSGGASLERIARLEEKVKSFKAQNSEN ASTANMLREQVAQLKQKGAP (SEQ ID NO:4), orASLERIARLEEKVKSFKAQNSENAST ANMLREQVAQLKQKGAP (SEQ ID NO:12).

Embodiment 120. The fusion protein of any one of embodiments 115 to 117wherein the c-jun domain comprises the amino acid sequenceSGASLERIARLEEKVKTLKAQNSEL ASTANMLREQVAQLKQKGAPSGGC (SEQ ID NO:2),SGASLERIARLEEKVKSFKAQN SENASTANMLREQVAQLKQKGAPSGGC (SEQ ID NO:5).

Embodiment 121. The fusion protein of any one of embodiments 115 to 117wherein the c-Fos domain comprises the amino acid sequenceASRELTDTLQAETDQLEDEKSALQTE IANLLKEKEKLEGAP (SEQ ID NO:3) orSGASRELTDTLQAETDQLEDEKSALQTEIANLL KEKEKLEGAP (SEQ NO:13).

Embodiment 122. The fusion protein of any one of embodiments 107 to 121wherein the fusion protein comprises a linker between the protein ofinterest and the ligand binding domain.

Embodiment 123. The fusion protein of embodiment 122 wherein the linkeris a Ser/Gly linker, a Poly-Asparagine linker, or a linker comprisingthe amino acid sequence AGSSAAGS GS (SEQ ID NO:17).

Embodiment 124. The fusion protein of embodiment 123 wherein thePoly-Asparagine linker comprises from about 8 to about 16 asparagineresidues.

Embodiment 125. The fusion protein of embodiment 123 wherein the Ser/Glylinker comprises GGSGGGSGGGSGGGSGGG (SEQ ID NO:18), GGSGGGSGGGSGGGSGGGSGGG (SEQ ID NO:19), GGSGGGSGGGSGGGSGGGSGGGSGGG (SEQ ID NO:20), SGGGGSGGGGSGGGG (SEQ ID NO:21). SGGGGSGGGGSGGGGSGGGG (SEQ ID NO:22), SGGGGSGGGGSGGGGSGGGGSGGGG (SEQ ID NO:23), SGGGS (SEQ ID NO:24), SGSG (SEQ IDNO:25), SGGGGS (SEQ ID NO:26), or SGSGG (SEQ ID NO:27).

Embodiment 126. The fusion protein of any one of embodiments 107 to 125wherein the protein of interest is a fragment of: a cytotoxic protein, amicrobicidal protein, a virucidal protein, a pro-apoptotic protein, athrombogenic protein, a complement activating protein, a Toll-LikeReceptor protein, a NOD2 receptor agonist protein, or an antibody orfragment thereof.

Embodiment 127. The fusion protein of embodiment 126 wherein: thecytotoxic protein is a bee melittin, a conotoxin, a cathelicidin, adefensin, a protegrin, or NK-lysin; the pro-apoptotic protein is prionprotein, a Bax-derived minimum poropeptide associated with the caspasecascade, or a pro-apoptotic peptide (KLAKLAK)₂ (SEQ ID NO:28); theinnate immune system stimulation protein is a pathogen-associatedmolecular pattern (PAMP) or a damage-associated molecular pattern(DAMP); the complement activating protein is a C3a fragment ofcomplement protein C3; the Toll-like Receptor (TLR) protein is a heatshock protein (hsp); the NOD2 receptor agonist protein is muramyldipeptide agonist; and the antibody fragment is an Fab, Fv, or scFv.

Embodiment 128. The fusion protein of any one of embodiments 107 to 125wherein the protein of interest is a fragment of: superfolder GFP(sfGFP), Renilla luciferase, murine dihydrofolate reductase (DHFR), S.cerevisiae ubiquitin, β-lactamase, or Herpes simplex virus type 1thymidine kinase.

Embodiment 129. The fusion protein of embodiment 128 wherein: thefragment of superfolder GFP (sfGFP) comprisesMRKGEELFTGVVPIINELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTIGKLIWPWPTINTILTYGVQCFARYPDHMKQHDEFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYI TADKQ (SEQID NO:29) or KNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK (SEQ ID NO:30); the fragmentof Renilla luciferase comprises MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAENAVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAWFELLNLPKKIIFVGHDWGACLAFITYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDIAIIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPREIPLVKGG (SEQ ID NO:31) or KPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKVKGLHFSQEDAPDEMGKYIKSFVERVLKNEQ (SEQ ID NO:32);the fragment of murine dihydrofolate reductase (DHFR) comprises aminoacids 1-105 or 106-186 thereof; the fragment of S. cerevisiae ubiquitincomprises amino acids 1-34 (MQIEVICTLTGKTITLEVESSDTIDNVKSKIQDKE: SEQ IDNO:33) or 35-76 (GIPPDQQ RLIFAGKQLEDGRTLSDYNIQKESTLIILVLRLRGG; SEQ IDNO:34) thereof; the fragment of β-lactamase comprises amino acids 25-197or 198-286 thereof; and the fragment of Herpes simplex virus type 1thymidine kinase comprises amino acids 1-265 or 266-376 thereof.

Embodiment 130. A composition or kit comprising a first fusion proteinof embodiment 107 and a second fusion protein of embodiment 107, whereinthe protein of interest of the first fusion protein and the protein ofinterest of the second fusion protein can dimerize or fold together.

Embodiment 131. The composition or kit of embodiment 130 wherein: thefirst fusion protein comprises a protein of interest fused to a ligandbinding domain for a small molecule ligand; the second fusion proteincomprises a protein of interest fused to a ligand binding domain for asmall molecule ligand.

Embodiment 132. The composition or kit of embodiment 131 wherein theligand binding domain of both the first fusion protein and the secondfusion protein are an FKBP domain or an FRB domain.

Embodiment 133. The composition or kit of embodiment 132 wherein theFKBP domain is a mutant FKBP domain.

Embodiment 133B. The fusion protein of embodiment 132 wherein the FKBPdomain comprises a C225, C22A, or C22V substitution, or wherein the FRBdomain comprises a C61S, C61A, or C61V substitution.

Embodiment 134. The composition or kit of embodiment 133 wherein themutant FKBP domain is the F36V FKBP mutant domain comprising the aminoacid sequence GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO:14) or MGVQVETISPGDGRTFPKRGQTCVVIIYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYG.ATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO:15).

Embodiment 135. The composition or kit of embodiment 130 wherein: thefirst fusion protein comprises a protein of interest fused to aninteractive protein domain; the second fusion protein comprises aprotein of interest fused to an interactive protein domain.

Embodiment 136. The composition or kit of embodiment 135 wherein theinteractive protein domain of both fusion proteins comprises less than100 amino acid residues.

Embodiment 137. The composition or kit of embodiment 135 wherein theinteractive protein domain of both fusion proteins is a leucine zipperdomain.

Embodiment 138. The composition or kit of embodiment 114 wherein theinteractive protein domain of the first fusion protein and the secondfusion protein is, independently, an NZ domain, a CZ domain, a c-jundomain, a c-fos domain, a c-myc domain, or a c-max domain.

Embodiment 139. The composition or kit of any one of embodiments 135 to138 wherein: the interactive protein domain of the first fusion proteinis fused to the N-terminus of the protein of interest, and theinteractive protein domain of the second fusion protein is fused to theN-terminus of the protein of interest; the interactive protein domain ofthe first fusion protein is fused to the C-terminus of the protein ofinterest, and the interactive protein domain of the second fusionprotein is fused to the C-terminus of the protein of interest; or theinteractive protein domain of one of the first fusion protein and secondfusion protein is fused to the N-terminus of the protein of interest,and the interactive protein domain of the other of the first fusionprotein and second fusion protein is fused to the C-terminus of theprotein of interest.

Embodiment 140. The composition or kit of any one of embodiments 135 to139 wherein the NZ domain comprises the amino acid sequenceALKKELQANKKELAQLKWELQ ALKKELAQ (SEQ ID NO:10), and the CZ domaincomprises the amino acid sequence EQLEKKLQALEKKLAQLEWKNQALEKKLAQ (SEQ IDNO:11).

Embodiment 141. The composition or kit of any one of embodiments 135 to139 wherein the c-jun domain comprises the amino acid sequenceCSGGASLERIARLEEKVKTL KAQNSELASTANMLREQVAQLKQKGAP (SEQ ID NO:1),CSGGASLERIARLEEKVKSFK AQNSENASTANTMLREQVAQLKQKG AP (SEQ ID NO:4), orASLERIARLEEKVKSFKAQ NSENASTANMLREQVAQLKQKGAP (SEQ ID NO:16).

Embodiment 142. The composition or kit of any one of embodiments 135 to139 wherein the c-jun domain comprises the amino acid sequenceSGASLERIARLEEKVKTLK AQNSELASTANMLREQVAQLKQKGAPSGGC (SEQ ID NO:2) orSGASLERIARLEEKV KSFKAQNSENASTANMLREQVAQLKQKGAPSGGC (SEQ ID NO:5).

Embodiment 143. The composition or kit of any one of embodiments 135 to139 wherein the c-Fos domain comprises the amino acid sequenceASRELTDTLQAETDQLEDE KSALQTEIANLLKEKEKLEGAP (SEQ ID NO:3) orSGASRELTDTLQAETDQLEDEKS ALQTEIANLLKEKEKLEGAP (SEQ ID NO:13).

Embodiment 144. The composition or kit of any one of embodiments 130 to143 wherein both the first fusion protein and second fusion proteincomprise a linker between the protein of interest and the ligand bindingdomain.

Embodiment 145. The composition or kit of embodiment 144 wherein eachlinker is, independently, a Ser/Gly linker, a Poly-Asparagine linker, ora linker comprising the amino acid sequence AGSSAAGSGS (SEQ ID NO:17).

Embodiment 146. The composition or kit of embodiment 145 wherein eachPoly-Asparagine linker, independently, comprises from about 8 to about16 asparagine residues.

Embodiment 147. The composition or kit of embodiment 145 wherein eachSer/Gly linker, independently, comprises GGSGGGSGGGSGGGSGGG (SEQ IDNO:18), GGSGG GSGGGSGGGSGGGSGGG (SEQ ID NO:19),GGSGGGSGGGSGGGSGGGSGGGSGGG (SEQ ID NO:20), SGGGGSGGGGSGGGG (SEQ IDNO:21), SGGGGSGGGGSGGGGSGG GG (SEQ ID NO:22), SGGGGSGGGGSGGGGSGGGGSGGGG(SEQ ID NO:23), SGGGS (SEQ ID NO:24), SGSG (SEQ ID NO:25), SGGGGS (SEQID NO:26), or SGSGG (SEQ ID NO:27).

Embodiment 148. The composition or kit of any one of embodiments 130 to147 wherein: the protein of interest of the first fusion protein is afirst fragment of a cytotoxic protein, and the protein of interest ofthe second fusion protein is a second fragment of a cytotoxic protein,wherein the first fragment of the cytotoxic protein and the secondfragment of the cytotoxic protein dimerize or fold together; the proteinof interest of the first fusion protein is a first fragment of amicrobicidal protein, and the protein of interest of the second fusionprotein is a second fragment of a microbicidal protein, wherein thefirst fragment of the microbicidal protein and the second fragment ofthe microbicidal protein dimerize or fold together; the protein ofinterest of the first fusion protein is a first fragment of a virucidalprotein, and the protein of interest of the second fusion protein is asecond fragment of a virucidal protein, wherein the first fragment ofthe virucidal protein and the second fragment of the virucidal proteindimerize or fold together; the protein of interest of the first fusionprotein is a first fragment of a pro-apoptotic protein, and the proteinof interest of the second fusion protein is a second fragment of apro-apoptotic protein, wherein the first fragment of the pro-apoptoticprotein and the second fragment of the pro-apoptotic protein dimerize orfold together; the protein of interest of the first fusion protein is afirst fragment of a thrombogenic protein, and the protein of interest ofthe second fusion protein is a second fragment of a thrombogenicprotein, wherein the first fragment of the thrombogenic protein and thesecond fragment of the thrombogenic protein dimerize or fold together;the protein of interest of the first fusion protein is a first fragmentof a complement activating protein, and the protein of interest of thesecond fusion protein is a second fragment of a complement activatingprotein, wherein the first fragment of the complement activating proteinand the second fragment of the complement activating protein dimerize orfold together; the protein of interest of the first fusion protein is afirst fragment of a Toll-Like Receptor protein, and the protein ofinterest of the second fusion protein is a second fragment of aToll-Like Receptor protein, wherein the first fragment of the Toll-LikeReceptor protein and the second fragment of the Toll-Like Receptorprotein dimerize or fold together; the protein of interest of the firstfusion protein is a first fragment of a NOD2 receptor agonist protein,and the protein of interest of the second fusion protein is a secondfragment of a NOD2 receptor agonist protein, wherein the first fragmentof the NOD2 receptor agonist protein and the second fragment of the NOD2receptor agonist protein dimerize or fold together; or the protein ofinterest of the first fusion protein is a first fragment of an antibodyor fragment thereof, and the protein of interest of the second fusionprotein is a second fragment of an antibody or fragment thereof, whereinthe first fragment of the antibody or fragment thereof and the secondfragment of the antibody or fragment thereof dimerize or fold together.

Embodiment 149. The composition or kit of embodiment 148 wherein: thecytotoxic protein is a bee melittin, a conotoxin, a cathelicidin, adefensin, a protegrin, or NK-lysin; the pro-apoptotic protein is prionprotein, a Bax-derived minimum poropeptide associated with the caspasecascade, or a pro-apoptotic peptide (KLAKLAK)₂ (SEQ ID NO:28); theinnate immune system stimulation protein is a pathogen-associatedmolecular pattern (PAMP) or a damage-associated molecular pattern(DAMP); the complement activating protein is a C3a fragment ofcomplement protein C3; the Toll-Like Receptor (TLR) protein is a heatshock protein (hsp); the NOD2 receptor agonist protein is muramyldipeptide agonist; and the antibody fragment is an Fab, Fv, or scFv.

Embodiment 150. The composition or kit of any one of embodiments 130 to147 wherein: the protein of interest of the first fusion protein is afirst fragment of superfolder GFP (sfGFP), and the protein of interestof the second fusion protein is a second fragment of sfGFP, wherein thefirst fragment of the sfGFP and the second fragment of the sfGFPdimerize or fold together; the protein of interest of the first fusionprotein is a first fragment of Renilla luciferase, and the protein ofinterest of the second fusion protein is a second fragment of Renillaluciferase, wherein the first fragment of the Renilla luciferase and thesecond fragment of the Renilla luciferase dimerize or fold together; theprotein of interest of the first fusion protein is a first fragment ofmurine dihydrofolate reductase (DHFR), and the protein of interest ofthe second fusion protein is a second fragment of DHFR, wherein thefirst fragment of the DHFR and the second fragment of the DHFR dimerizeor fold together; the protein of interest of the first fusion protein isa first fragment of S. cerevisiae ubiquitin, and the protein of interestof the second fusion protein is a second fragment of S. cerevisiaeubiquitin, wherein the first fragment of the S. cerevisiae ubiquitin andthe second fragment of the S. cerevisiae ubiquitin dimerize or foldtogether; the protein of interest of the first fusion protein is a firstfragment of β-lactamase, and the protein of interest of the secondfusion protein is a second fragment of β-lactamase, wherein the firstfragment of the β-lactamase and the second fragment of the β-lactamasedimerize or fold together; or the protein of interest of the firstfusion protein is a first fragment of Herpes simplex virus type 1thymidine kinase, and the protein of interest of the second fusionprotein is a second fragment of Herpes simplex virus type 1 thymidinekinase, wherein the first fragment of the Herpes simplex virus type 1thymidine kinase and the second fragment of the Herpes simplex virustype 1 thymidine kinase dimerize or fold together.

Embodiment 151. The composition or kit of embodiment 150 wherein: thefirst fragment or second fragment of sfGFP comprises the amino acidsequence MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQ (SEQ ID NO:29), and the other of the firstfragment or second fragment of sfGFP comprises the amino acid sequenceKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVITAAGITHGMD ELYK (SEQ IDNO:30); the first fragment or second fragment of Renilla luciferasecomprises the amino acid sequenceMASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAENAVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAWFELLNLPKKIIENGHDWGACLAPHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDIALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWP REIPLVKGG(SEQ ID NO:31), and the other of the first fragment or second fragmentof Renilla luciferase comprises the amino acid sequenceKPDVVQIVRNYNAYLRASDDLPKWIFIESDPGFFSNAIVEGAKKFPNTEFVKVICGLHESQEDAPDEMGKYIKSFVERVLKNEQ (SEQ ID NO:32); thefirst fragment or second fragment of DHFR comprises amino acids 1-105thereof, and the other of the first fragment or second fragment of DHFRcomprises amino acids 106-186 thereof; the first fragment or secondfragment of S. cerevisiae ubiquitin comprises the amino acid sequenceMQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKE (SEQ ID NO:33), and the other of thefirst fragment or second fragment of S. cerevisiae ubiquitin comprisesthe amino acid sequence GIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG (SEQID NO:34); the first fragment or second fragment of β-lactamasecomprises amino acids 25-197 thereof, and the other of the firstfragment or second fragment of β-lactamase comprises amino acids 198-286thereof; and the first fragment or second fragment of Herpes simplexvirus type 1 thymidine kinase comprises amino acids 1-265 thereof, andthe other of the first fragment or second fragment of Herpes simplexvirus type 1 thymidine kinase comprises amino acids 266-376 thereof.

Embodiment 152. A composition or kit comprising: a first haplomer-ligandcomplex of any one of embodiments 1, 2, or 3 to 10; a secondhaplomer-ligand complex of any one of embodiments 1, 2, or 3 to 10; afirst fusion protein of any one of embodiments 107 to 111 or 122 to 129;and a second fusion protein of any one of embodiments 107 to 111 or 122to 129; wherein the ligand of the first haplomer-ligand complex islinked to the 5′ terminus of the polynucleotide of the firsthaplomer-ligand complex; wherein the ligand of the secondhaplomer-ligand complex is linked to the 3′ terminus of thepolynucleotide of the second haplomer-ligand complex; wherein thepolynucleotide of the first haplomer-ligand complex is substantiallycomplementary to a target nucleic acid molecule; wherein thepolynucleotide of the second haplomer-ligand complex is substantiallycomplementary to the target nucleic acid molecule at a site in spatialproximity to the polynucleotide of the first haplomer-ligand complex;wherein the ligand of the first haplomer-ligand complex and the ligandbinding domain of the first fusion protein can interact; wherein theligand of the second haplomer-ligand complex and the ligand bindingdomain of the second fusion protein can interact; and wherein thefragment of the protein of interest of the first fusion protein and thefragment of the protein of interest of the second fusion protein candimerize or fold together.

Embodiment 153. A composition or kit comprising: a first haplomer-ligandcomplex of any one of embodiments 1, 2, or 11 to 18; a secondhaplomer-ligand complex of any one of embodiments 1, 2, or 11 to 18; afirst fusion protein of any one of embodiments 107 or 112 to 129; and asecond fusion protein of any one of embodiments 107 or 112 to 129;wherein the ligand of the first haplomer-ligand complex is linked to the5′ terminus of the polynucleotide of the first haplomer-ligand complex;wherein the ligand of the second haplomer-ligand complex is linked tothe 3′ terminus of the polynucleotide of the second haplomer-ligandcomplex; wherein the polynucleotide of the first haplomer-ligand complexis substantially complementary to a target nucleic acid molecule;wherein the polynucleotide of the second haplomer-ligand complex issubstantially complementary to the target nucleic acid molecule at asite in spatial proximity to the polynucleotide of the firsthaplomer-ligand complex; wherein the ligand of the first haplomer-ligandcomplex and the ligand binding domain of the first fusion protein caninteract; wherein the ligand of the second haplomer-ligand complex andthe ligand binding domain of the second fusion protein can interact; andwherein the fragment of the protein of interest of the first fusionprotein and the fragment of the protein of interest of the second fusionprotein can dimerize or fold together.

Embodiment 154. A composition or kit comprising: a first bottlehaplomer-ligand complex of any one of embodiments 44 to 65; a secondhaplomer-ligand complex of any one of embodiments 1, 2, or 3 to 10,wherein the second haplomer-ligand complex comprises a nucleotideportion that is substantially complementary to the stem portion of thebottle haplomer-ligand complex that is linked to the ligand of thebottle haplomer-ligand complex; a first fusion protein of any one ofembodiments 107 to 111 or 122 to 129; and a second fusion protein of anyone of embodiments 107 to 111 or 122 to 129; wherein the ligand of thefirst bottle haplomer-ligand complex and the ligand binding domain ofthe first fusion protein can interact; wherein the ligand of the secondhaplomer-ligand complex and the ligand binding domain of the secondfusion protein can interact; and wherein the fragment of the protein ofinterest of the first fusion protein and the fragment of the protein ofinterest of the second fusion protein can dimerize or fold together.

Embodiment 155. A composition or kit comprising: a first bottlehaplomer-ligand complex of any one of embodiments 44 to 57 or 66 to 73;a second haplomer-ligand complex of any one of embodiments 1, 2, or 11to 18, wherein the second haplomer-ligand complex comprises a nucleotideportion that is substantially complementary to the stem portion of thebottle haplomer-ligand complex that is linked to the ligand of thebottle haplomer-ligand complex; a first fusion protein of any one ofembodiments 107 or 112 to 129; and a second fusion protein of any one ofembodiments 107 or 112 to 129; wherein the ligand of the first bottlehaplomer-ligand complex and the ligand binding domain of the firstfusion protein can interact; wherein the ligand of the secondhaplomer-ligand complex and the ligand binding domain of the secondfusion protein can interact; and wherein the fragment of the protein ofinterest of the first fusion protein and the fragment of the protein ofinterest of the second fusion protein can dimerize or fold together.

Embodiment 156. A method for the directed assembly of a proteincomprising: contacting a target nucleic acid molecule with a firsthaplomer-ligand complex of any one of embodiments 1, 2, or 3 to 10;contacting the target nucleic acid with a second haplomer-ligand complexof any one of embodiments 1, 2, or 3 to 10; contacting the firsthaplomer-ligand complex with a first fusion protein of any one ofembodiments 107 to 111 or 122 to 129; and contacting the secondhaplomer-ligand complex with a second fusion protein of any one ofembodiments 107 to 111 or 122 to 129; wherein the ligand of the firsthaplomer-ligand complex is linked to the 5′ terminus of thepolynucleotide of the first haplomer-ligand complex; wherein the ligandof the second haplomer-ligand complex is linked to the 3′ terminus ofthe polynucleotide of the second haplomer-ligand complex; wherein thepolynucleotide of the first haplomer-ligand complex is substantiallycomplementary to a target nucleic acid molecule; wherein thepolynucleotide of the second haplomer-ligand complex is substantiallycomplementary to the target nucleic acid molecule at a site in spatialproximity to the polynucleotide of the first haplomer-ligand complex;wherein the ligand of the first haplomer-ligand complex and the ligandbinding domain of the first fusion protein can interact; and wherein theligand of the second haplomer-ligand complex and the ligand bindingdomain of the second fusion protein can interact; thereby resulting inthe folding or dimerization of the fragment of the protein of interestof the first fusion protein with the fragment of the protein of interestof the second fusion protein.

Embodiment 157. A method for the directed assembly of a proteincomprising: contacting a target nucleic acid molecule with a firsthaplomer-ligand complex of any one of embodiments 1, 2, or 11 to 18;contacting the target nucleic acid with a second haplomer-ligand complexof any one of embodiments 1, 2, or 11 to 18; contacting the firsthaplomer-ligand complex with a first fusion protein of any one ofembodiments 107 or 112 to 129; and contacting the second haplomer-ligandcomplex with a second fusion protein of any one of embodiments 107 or112 to 129; wherein the ligand of the first haplomer-ligand complex islinked to the 5′ terminus of the polynucleotide of the firsthaplomer-ligand complex; wherein the ligand of the secondhaplomer-ligand complex is linked to the 3′ terminus of thepolynucleotide of the second haplomer-ligand complex; wherein thepolynucleotide of the first haplomer-ligand complex is substantiallycomplementary to a target nucleic acid molecule; wherein thepolynucleotide of the second haplomer-ligand complex is substantiallycomplementary to the target nucleic acid molecule at a site in spatialproximity to the polynucleotide of the first haplomer-ligand complex;wherein the ligand of the first haplomer-ligand complex and the ligandbinding domain of the first fusion protein can interact; and wherein theligand of the second haplomer-ligand complex and the ligand bindingdomain of the second fusion protein can interact; thereby resulting inthe folding or dimerization of the fragment of the protein of interestof the first fusion protein with the fragment of the protein of interestof the second fusion protein.

Embodiment 158. The method of embodiment 156 or embodiment 157 whereinthe polynucleotide of the first haplomer-ligand complex is complementaryto the polynucleotide of the second haplomer-ligand complex.

Embodiment 159. The method of embodiment 156 or embodiment 157 whereinthe polynucleotide of the first haplomer-ligand complex binds to thetarget nucleic acid molecule in spatial proximity to the binding of thepolynucleotide of the second haplomer-ligand complex to the targetnucleic acid molecule.

Embodiment 160. The method of embodiment 156 or embodiment wherein: theligand of the first haplomer-ligand complex is linked to the 5′ terminusof the polynucleotide of the first haplomer-ligand complex, and thepolynucleotide of the first haplomer-ligand complex is complementary toa portion of the nucleic acid target 5′ adjacent to a stem-loopstructure; and the ligand of the second haplomer-ligand complex islinked to the 3′ terminus of the polynucleotide of the secondhaplomer-ligand complex, and the polynucleotide of the secondhaplomer-ligand complex is complementary to a portion of the nucleicacid target 3′ adjacent to the stem-loop structure.

Embodiment 161. The method of embodiment 156 or embodiment 157 wherein:the ligand of the first haplomer-ligand complex is linked to the 3′terminus of the polynucleotide of the first haplomer-ligand complex, andthe polynucleotide of the first haplomer-ligand complex is complementaryto a 5′ portion of a loop structure of a stem-loop structure of thenucleic acid target, wherein the 5′ portion of the loop structure isadjacent to the stem region of the stem-loop structure; and the ligandof the second haplomer-ligand complex is linked to the 5′ terminus ofthe polynucleotide of the second haplomer-ligand complex, and thepolynucleotide of the second haplomer-ligand complex is complementary toa 3′ portion of the loop structure of the stem-loop structure of thenucleic acid target, wherein the 3′ portion of the loop structure isadjacent to the stem region of the stem-loop structure.

Embodiment 162. A method for the directed assembly of a proteincomprising: contacting a target nucleic acid molecule with a complexformed by the interaction of a first haplomer-ligand complex of any oneof embodiments 1, 2, or 3 to 10 with a first fusion protein of any oneof embodiments 107 to 111 or 122 to 129, wherein the ligand of the firsthaplomer-ligand complex is linked to the 5′ terminus of the polynucleotide of the first haplomer-ligand complex, and wherein the ligandof the first haplomer-ligand complex interacts with the ligand bindingdomain of the first fusion protein; and contacting the target nucleicacid molecule with a complex formed by the interaction of a secondhaplomer-ligand complex of any one of embodiments 1, 2, or 3 to 10 witha second fusion protein of any one of embodiments 107 to 111 or 122 to129, wherein the ligand of the second haplomer-ligand complex is linkedto the 5′ terminus of the polynucleotide of the second haplomer-ligandcomplex, and wherein the ligand of the second haplomer-ligand complexligand binding domain of the second fusion protein; thereby resulting inthe folding or dimerization of the fragment of the protein of interestof the first fusion protein with the fragment of the protein of interestof the second fusion protein.

Embodiment 163. A method for the directed assembly of a proteincomprising: contacting a target nucleic acid molecule with a complexformed by the interaction of a first haplomer-ligand complex of any oneof embodiments 1, 2, or 11 to 18 with a first fusion protein of any oneof embodiments 107 or 112 to 129, wherein the ligand of the firsthaplomer-ligand complex is linked to the 5′ terminus of thepolynucleotide of the first haplomer-ligand complex, and wherein theligand of the first haplomer-ligand complex interacts with the ligandbinding domain of the first fusion protein; and contacting the targetnucleic acid molecule with a complex formed by the interaction of asecond haplomer-ligand complex of any one of embodiments 1, 2, or 11 to18 with a second fusion protein of any one of embodiments 107 or 112 to129, wherein the ligand of the second haplomer-ligand complex is linkedto the 5′ terminus of the polynucleotide of the second haplomer-ligandcomplex, and wherein the ligand of the second haplomer-ligand complexinteracts with the ligand binding domain of the second fusion protein;thereby resulting in the folding or dimerization of the fragment of theprotein of interest of the first fusion protein with the fragment of theprotein of interest of the second fusion protein.

Embodiment 164. The method of embodiment 162 or embodiment 163 whereinthe polynucleotide of the first haplomer-ligand complex is complementaryto the polynucleotide of the second haplomer-ligand complex.

Embodiment 165. The method of embodiment 162 or embodiment 163 whereinthe polynucleotide of the first haplomer-ligand complex binds to thetarget nucleic acid molecule in spatial proximity to the binding of thepolynucleotide of the second haplomer-ligand complex to the targetnucleic acid molecule.

Embodiment 166. The method of embodiment 162 or embodiment 163 wherein:the ligand of the first haplomer-ligand complex is linked to the 5′terminus of the polynucleotide of the first haplomer-ligand complex, andthe polynucleotide of the first haplomer-ligand complex is complementaryto a portion of the nucleic acid target 5′ adjacent to a stem-loopstructure; and the ligand of the second haplomer-ligand complex islinked to the 3′ terminus of the polynucleotide of the secondhaplomer-ligand complex, and the polynucleotide of the secondhaplomer-ligand complex is complementary to a portion of the nucleicacid target 3′ adjacent to the stem-loop structure.

Embodiment 167. The method of embodiment 162 or embodiment 163 wherein:the ligand of the first haplomer-ligand complex is linked to the 3′terminus of the polynucleotide of the first haplomer-ligand complex, andthe polynucleotide of the first haplomer-ligand complex is complementaryto a 5′ portion of a loop structure of a stem-loop structure of thenucleic acid target, wherein the 5′ portion of the loop structure isadjacent to the stem region of the stem-loop structure; and the ligandof the second haplomer-ligand complex is linked to the 5′ terminus ofthe polynucleotide of the second haplomer-ligand complex, and thepolynucleotide of the second haplomer-ligand complex is complementary toa 3′ portion of the loop structure of the stem-loop structure of thenucleic acid target, wherein the 3′ portion of the loop structure isadjacent to the stem region of the stein-loop structure.

Embodiment 168. A method for the directed assembly of a proteincomprising: contacting a target nucleic acid molecule with a bottlehaplomer-ligand complex of any one of embodiments 44 to 57 or 58 tocontacting the target nucleic acid with a second haplomer-ligand complexof any one of embodiments 1, 2, or 3 to 10 , wherein the secondhaplomer-ligand complex comprises a nucleotide portion that issubstantially complementary to the stem portion of the bottlehaplomer-ligand complex that is linked to the ligand of the bottlehaplomer-ligand complex; contacting the bottle haplomer-ligand complexwith a first fusion protein of any one of embodiments 107 to 111 or 122to 129, wherein the ligand of the bottle haplomer-ligand complex and theligand binding domain of the first fusion protein can interact; andcontacting the second haplomer-ligand complex with a second fusionprotein of any one of embodiments 107 to 111 or 122 to 129, wherein theligand of the second haplomer-ligand complex and the ligand bindingdomain of the second fusion protein can interact; thereby resulting inthe folding or dimerization of the fragment of the protein of interestof the first fusion protein with the fragment of the protein of interestof the second fusion protein.

Embodiment 169. A method for the directed assembly of a proteincomprising: contacting a target nucleic acid molecule with a bottlehaplomer-ligand complex of any one of embodiments44 to 57 or 66 to 73;contacting the target nucleic acid with a second haplomer-ligand complexof any one of embodiments 1, 2, or 11 to 18, wherein the secondhaplomer-ligand complex comprises a nucleotide portion that issubstantially complementary to the stem portion of the bottlehaplomer-ligand complex that is linked to the ligand of the bottlehaplomer-ligand complex; contacting the bottle haplomer-ligand complexwith a first fusion protein of any one of embodiments 107 or 112 to 129,wherein the ligand of the bottle haplomer-ligand complex and the ligandbinding domain of the first fusion protein can interact; and contactingthe second haplomer-ligand complex with a second fusion protein of anyone of embodiments 107 or 112 to 129, wherein the ligand of the secondhaplomer-ligand complex and the ligand binding domain of the secondfusion protein can interact; thereby resulting in the folding ordimerization of the fragment of the protein of interest of the firstfusion protein with the fragment of the protein of interest of thesecond fusion protein.

Embodiment 170. A method for the directed assembly of a proteincomprising: contacting a target nucleic acid molecule with a bottlehaplomer-ligand complex of any one of embodiments 44 to 65; contactingthe target nucleic acid molecule with a second haplomer-ligand complexof any one of embodiments 1, 2, or 3 to 10, wherein the secondhaplomer-ligand complex comprises a nucleotide portion that issubstantially complementary to the stem portion of the bottlehaplomer-ligand complex that is linked to the ligand of the bottlehaplomer-ligand complex; contacting the bottle haplomer-ligand complexwith a first fusion protein of any one of embodiments 107 to 111 or 122to 129, wherein the ligand of the bottle haplomer-ligand complex and theligand binding domain of the first fusion protein can interact; andcontacting the second haplomer-ligand complex with a second fusionprotein of any one of embodiments 107 to 111 or 122 to 129, wherein theligand of the second haplomer-ligand complex and the ligand bindingdomain of the second fusion protein can interact; thereby resulting inthe folding or dimerization of the fragment of the protein of interestof the first fusion protein with the fragment of the protein of interestof the second fusion protein.

Embodiment 171. A method for the directed assembly of a proteincomprising: contacting a target nucleic acid molecule with a bottlehaplomer-ligand complex of any one of embodiments 44 to 57 or 66 to 73;contacting the target nucleic acid molecule with a secondhaplomer-ligand complex of any one of embodiments 1, 2, or 11 to 18,wherein the second haplomer-ligand complex comprises a nucleotideportion that is substantially complementary to the stem portion of thebottle haplomer-ligand complex that is linked to the ligand of thebottle haplomer-ligand complex; contacting the bottle haplomer-ligandcomplex with a first fusion protein of any one of embodiments 107 or 112to 129, wherein the ligand of the bottle haplomer-ligand complex and theligand binding domain of the first fusion protein can interact; andcontacting the second haplomer-ligand complex with a second fusionprotein of any one of embodiments 107 or 112 to 129, wherein the ligandof the second haplomer-ligand complex and the ligand binding domain ofthe second fusion protein can interact; thereby resulting in the foldingor dimerization of the fragment of the protein of interest of the firstfusion protein with the fragment of the protein of interest of thesecond fusion protein.

In order that the subject matter disclosed herein may be moreefficiently understood, examples are provided below. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting the claimed subject matter in anymanner.

EXAMPLES Example 1, Expression of c-Jun Leucine Zipper Domains via theMaltose-Binding Protein System, and Conjugation with SpecificOligonucleotides to Form an LD-TAPER Haplomer

c-Jun fragments for conjugation with oligonucleotides to form smallprotein interactive domain LD-TAPER haplomers were expressed as fusionproteins in the maltose-binding protein (MBP) system, before releasefrom the MBP fusion protein with enterokinase.

Coding sequences for each c-Jun segment (bearing either an N-terminalcysteine or a C-terminal cysteine, as above) were equipped with anenterokinase recognition signal (codons for DDDDK; SEQ ID NO:35), suchthat after expression, the C-terminal MBP fragments could be cleavedfrom the maltose binding carrier protein. Assembled sequences werecloned between XmnI and SbfI sites of pMALc5x (New England Biolabs), andthe structure of candidate clones confirmed by sequencing. Verifiedclones were transformed into the strain NEB-express (New EnglandBiolabs), and propagated in liquid culture (50 ml) under short-termgrowth conditions at 37° C. for 1.5 hours, before induction with 300 μMIPTG for a further 2 hours. Samples (“direct lysates”; 200 μl ) weretaken, pelleted in 1.5 ml tubes at 1000 x g, washed once with :200 μl of1X PBS, and re-suspended in 50 μl of PBS. The remainder of the 50 mlgrowths were pelleted. (10 minutes/3000 rpm in a Sorvall benchtopcentrifuge), and re-suspended in 2.0 ml Eppendorf tubes in 1.5 ml ofice-cold maltose-binding protein system column buffer (MC-buffer: 20 mMTris (pH 7.4), 200 mM NaCl, 1 mM EDTA, and 1 DTT) also containing 1%protease inhibitor cocktail (Sigma P3840). Cell suspensions were thensonicated (6×5 second pulses, 5-setting, Branson 450 Sonifier, withchilling between each sonication round), centrifuged for 5 minutes at14000 rpm (benchtop microfuge), and the supernatants were transferred toa fresh tube.

Polypeptides expressed as fusions with maltose-binding protein wereaffinity purified on amylose magnetic beads (A-MBs; New EnglandBiolabs). Suitable samples of A-MBs (usually equivalent to 250 μl of theoriginal slurries per 1 ml of supernatant) were washed twice with 1 mlof cold MC-buffer (using magnetic separation to pull down the A-MBs),and re-suspended in the original volume. Sonicated supernatants frominduced plasmid cultures were mixed with the A-MBs for 1 hour at 4° C.,with frequent tube inversion to re-suspend the beads. The supernatantswere then magnetically removed, and the beads washed four times with 0.5ml of cold MC-buffer before resuspension in 150 μl of the samebuffer/250 μl of original beads.

Bound proteins were eluted with final concentration of 10 mM maltose.The elution and enterokinase treatments were performed simultaneously bythe addition of 2 mM calcium chloride to the MC-buffer containing themaltose. After 3 hours of elution/digestion, supernatants weremagnetically separated, the pellets treated with 75 μl of MC-buffer, andthe soluble phase combined with the first supernatant. A portion of thisproduct was then subjected to an agarose affinity treatment to removeenterokinase (EMD-Millipore), and the non-binding flow-through retained.Samples were analyzed initially on a TGX any-kD gel (BioRad), andstained with SYPRO-Ruby (ThermoFisher). Expression of both c-Jun MBPfusions as soluble proteins occurred at very high levels (see, lanes 1and 2, FIG. 23 ), considerably in excess of the maximal retainable bythe amount of A-MBs used (see, lanes 1 and 2 vs. lanes 3 and 4, FIG. 23). Enterokinase cleavage of co-eluted fusion protein went to completionin the case of the C-terminal cysteine c-Jun fragment (see, lane 6, FIG.23 ), and was approximately 80% complete for the N-terminal cysteinefusion (see, lane 5, FIG. 23 ). Recovery of the products followingenterokinase removal was on the order of 75% (see, lanes 7 and 8 vs.lanes 5 and 6, FIG. 23 ). In both cases, enterokinase cleavage producedthe expected MBP cleavage band, but under conditions used, the smallc-Jun bands were not visible, Subsequently, samples of cleaved productswere run on a 16% Tris-Tricine gel (ThermoFisher) and stained withFlamingo stain (BioRad), after which the expected 5.0 and 5.2 kD bandsfor the N-terminal and C-terminal cysteine c-Jun fragments(respectively) could he visualized (see, lanes 9-12, FIG. 23 ),

Since both c-Jun fragments lack internal cysteines, they are readilyamenable to conjugation with thiol-labeled oligonucleotides viabifunctional maleimide reagents. The conjugation process usingbis-maleimide linkers is performed in two stages. Initially,oligonucleotides bearing a 5′ or 3′ terminal disulfide modification aretreated with 100-fold molar excess of TCEP for at least 4 hours at 25°C., and then desalted into 10 mM Tris (pH 7.4) to remove the TCEP andlow-molecular weight products. The resulting —SH oligonucleotides arethen treated with a molar excess (500-fold) of1,8-bis(maleimido)diethylene glycol (BMP2, Sigma) in sodium phosphate buffer pH 7.1 for 4hours at 25° C. The preparations are then desalted once more to removeexcess BMP2. Samples of the modified oligonucleotides are run on 8 Murea gels to examine the success of the process, in comparison to theoriginal —SS— oligonucleotides and the corresponding derived —SHoligonucleotides.

The second stage uses the BMP2-derivatized oligonucleotide to cross-linkto the c-Jun fragments with N- and C-terminal cysteine residues. Beforetreatment, the cleaved fragment-MBP preparations are treated with a10-fold molar excess of TCEP to ensure that the cysteine —SH groups arereduced, and then desalted into phosphate buffer (p171 7.1) with 100 mMsodium chloride.

Fragments are incubated in the same buffer with a large molar excess ofBMP2-derivatized oligonucleotide to drive the reaction, for 4 hours at25° C. Excess oligonucleotides (bearing unreacted maleimide groups) arethen removed by treatment with sulfhydryl magnetic bead (Bioclone).Polypeptide conjugates are then dialyzed against PBSM and stored in 50%glycerol at −20° C.

Example 2: Use of c-Tun Haplomers with c-Fos Polypeptide Fusions forSplit-Protein LD-TAPER (Prophetic)

Where c-Jun zipper fragments are used to implement LD-TAPER (byconstructing haplomers with 5′ and 3′ c-Jun Lags; see, Example 1), afterhybridization of haplomers to target templates, the second stage usesfusion proteins with complementary c-Fos zippers. Thus, for theapplication of split-protein technology with small interactive proteindomains to LD-TAPER, suitable protein fragments are expressed as N- orC-terminal c-Fos fusions. Superfolder GFP (sfGFP) and Renilla luciferasefragments are used.

Locked TAPER (see, FIG. 13 ) is used with c-Jun tags, instead ofsmall-molecule ligands (see, FIG. 24 ). The 5′ end of the first bottlehaplomer is conjugated by thiol-maleimide chemistry with the c-Junfragment with an N-terminal cysteine, and the 3′ end of the secondhaplomer is conjugated with the c-Jun fragment with a C-terminalcysteine (sequences as above). With this arrangement, the zippers arealigned in an antiparallel orientation following “unlocking” of theinitial first bottle haplomer anti-target loop portion in the presenceof specific template (see, FIG. 24 ).

Fos-fusion protein sequences are as follows (c-Fos sequence are in bold,including helix boundary signals; double underlined segments indicateserine-glycine linkers): N-terminal sfGFP/C-terminal Fos:

(SEQ ID NO: 36) MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQC FSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAVVKFEGDTIVNRIELKGTDFKEDGNILGHK LEYNFNSHNVYITADKQGGSGGGSGGGSGGGSGGGASRELTDTLQAETDQLEDEKSALQTEIANLLKEKE KLEGAP*;N-terminal Fos/C-terminal sfGFP: (SEQ ID NO: 37)SGASRELTDTLQAETDQLEDEKSALQTEIANLLKE KEKLEGAP GGSGGGSGGGSGGGSGGGKNGIKANFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYL STQTVLSKDPNEKRDHMVLHEYVNAAGITLGMDELYK*; N-terminal Renilla/C-terminal Fos: (SEQ ID NO: 38)MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFIN YYDSEKHAENAVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAWF ELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDIALIKSEEGEKMV LENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPREIPLVKGGGGSGGGSGGGSGGGSG GG ASRELTDTLQAETDQLEDEKSALQTEIANLLKEKEKLEGAP*; N-terminal Fos/C-terminal Renilla: (SEQ ID NO: 39)SGASRELTDTLQAETDQLEDEKSALQTEIANLLK EKEKLEGAP GGSGGGSGGGSGGGSGGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKK FPNTEFVKVKGLHFSQEDAPDEMGKYIKSFVERVLKNEQ*.

For expression purposes, all protein codons are optimized for E. coilK12, All proteins are expressed as maltose-binding protein (MBP) fusions(see, Example 1), and released from the MBP carrier by treatment withenterokinase.

In the initial experimental approach “A”, to implement the experimentalprocess, the c-Jun first bottle haplomer is incubated with specifictarget template (complementary to its anti-target loop portion),allowing “unlocking” of this first bottle haplomer, and exposure of thehybridization site for the second haplomer. After duplex formation withthe second haplomer, the preparations of c-Fos fusions of sfGFP andRenilla N- and C-terminal fragments (as above) are added. This allowsthe second stage of the LD TAPER process to proceed, with c-Fos:c-Junzippers pairing, and thereby juxtaposing the polypeptide split-proteinpair for mature folding (see, FIG. 25 ). The antiparallel orientation ofthe haplomer c-Jun tags (see, FIG. 24 ) facilitates the positioning andorientation of the polypeptides (see, FIG. 25 ).

The sfGFP signal is fluorescence at the same emission maximum as forfluorescein, and is monitored by means of a spectrophotometer withfluorescent reading facility (Tecan). The enzymatic activity of Renillaluciferase is assessed by means of commercial kits for this enzyme(Promega), using coelenterazine substrate, and purified Renillaluciferase (RayBiotech) as a positive control. Luminescence isquantified by means of a standard luminometer (Berthold).

In a dose-response experimental design, equimolar amounts of sfGFP N-and C-terminal Fos-fusions are mixed with first-stage haplomers“unlocked” in the presence of specific target template as above. Ratiosof the N- and C-terminal Fos fusions to haplomer range from 1:1 to 10:1.Analogous experiments are established with Renilla N- and C-terminalFos-fusions. After a 16 hour incubation at 25° C., reporter signals areassayed as appropriate for both sfGFP and Renilla luciferase.

Since homoligand LD-TAPER for split-protein folding has an efficiencylimitation if the haplomer/template hybridizations are performed as thefirst step (as shown with Equations 4.1, 4.2), in addition toexperimental approach “A,” a second (“B”) protocol is also followed, inaccord with Equations 5.1 and 5.2. Here the locked-TAPER first bottlehaplomer bearing a c-Jun tag (see, FIG. 24 ) is hybridized to templateas before, but then treated with the N-terminal sfGFP or RenillaC-terminal Fos fusions. In a separate reaction, the second haplomer istreated with the C-terminal sfGFP or Refill N-terminal fusions. After 2hours at 25° C., each pre-assembled c-Junhaplomer/template-Fos-polypeptide is mixed in equimolar amounts. After a16 hour incubation at 25° C., reporter signals are assayed asappropriate for both sfGFP and Renilla luciferase.

For both experimental designs “A” and “B”, comparable time-courseexperiments are also performed after the two-stage assembly of allLD-TAPER components has been completed. In protocol “A”, thiscorresponds to the initial hybridizations for both Jun-haplomers;followed by the addition of the split-protein Fos-polypeptides; forprotocol “B”, this corresponds to the separate pre-assembly ofJun-haplomers with specific split-protein Fos-polypeptides, followed bycombination of the two haplomeric complexes. For each arrangement,assayable samples taken at a series of time points: 15, 30, 45, and 60minutes: and 1, 2, 4, 6, 8, and 16 hours.

Specificity of the template-mediated LD-TAPER assembly may bedemonstrated by the use of a blocking oligonucleotide that correspondsto the same sequence as the second haplomer, but lacking any appendedtag. A molar excess of such an oligonucleotide effectively inhibits theassembly reaction, whereas the assembly process is unaffected by excessoligonucleotide of the same length but with scrambled sequence.

Example 3: Use of Oligonucleotide Conjugates with Mutant FKBP-BindingMonovalent Compounds in LD-TAPER for Split-Protein LD-TAPER (Prophetic)

This Example describes the derivatization of amino-labeledoligonucleotides with mutant FKBP-binding monovalent compounds, andtheir application in LD-TAPER for small-molecule ligand-directedsplit-protein folding. Compounds, such as FKM-NHS (see, FIG. 6 ), aresolubilized in DMSO to 10 mM and incubated in a 100-fold molar excess insodium phosphate buffer (pH 7.4) with oligonucleotides to be used ashaplomers, where either the 5′ or 3′ ends of the oligonucleotides aremodified with a commercially available aminolinker. After 2 hours at 25°C., excess unreacted compound is removed by desalting columns, afterwhich the resulting FKM-haplomers are stored in (10/1.0) TE buffer. Thesuccess of the formation of the oligonucleotide:FKM adducts is confirmedby testing product mobility on 15% 8 M urea gels, relative to untreatedparental oligonucleotide.

Haplomers bearing FKM as small-molecule ligands are then amenable tosplit-protein studies. In this Example, Locked TAPER is used, asdepicted in FIG. 13 . Polypeptide split-protein targets are rendered asfusion proteins with the F36V mutant of FKBP-1A. For the application ofsplit-protein technology with small molecule ligands to LD-TAPER,suitable protein fragments are expressed as N- or C-terminal mutant FKBP(F36V) fusions. Superfolder GFP (sfGFP) and Renilla luciferase fragmentsare used. For haplomer construction, Locked TAPER (see, FIG. 13 ) isused with FKM serving as the small molecule ligand (see, FIG. 26 ), withits conjugation to the haplomeric oligonucleotides performed asdescribed above.

Mutant FKBP (mFKBP)-fusion protein sequences are as follows (FKBPsequence is in bold, except for the F36V mutation, in small letters;double underlined segments indicate serine-glycine linkers): N-terminalsfGFP/C-terminal mFKBP:

MSKGEELFTGVVPILVELDGDVNG (SEQ ID NO: 40)HKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPT LVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAVVKFEGDTLVNRIELKGTD FKEDGNILGHKLEYNFNSHNVYITADKQGGSGGGSGGGSGGGSGGG GVQVETISPGDGRTFPKRGQTCVV HYTGMLEDGKKvDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPH ATLVFDVELLKLE*;N-terminal mFKBP/C-terminal sfGFP: (SEQ ID NO: 41)MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDG KKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL LKLE GGSGGGSGGGSGGGSGGGKNGIKANFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQT VLSKDPNEKRDHMVLHEYVNAAGITLGMDELYK*;N-terminal Renilla/C-terminal mFKBP: (SEQ ID NO: 42)MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFIN YYDSEKHAENAVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAWF ELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDIALIKSEEGEKMV LENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPREIPLVKGGGGSGGGSGGGSGGGSG GG GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE*; N-terminal mFKBP/C-terminal Renilla: (SEQ ID NO: 43)MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGK KvDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL KLE GGSGGGSGGGSGGGSGGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEF VKVKGLHFSQEDAPDEMGKYIKSFVERVLKNEQ*.

For expression purposes, all protein codons are optimized for E. coilK12, All proteins are expressed as maltose-binding protein (MBP) fusions(see, Example 1), and released from the MBP carrier by treatment withenterokinase.

In the initial experimental approach “A”, to implement the experimentalprocess, the FKM-first bottle haplomer is incubated with specific targettemplate (complementary to its loop region), allowing “unlocking” ofthis first haplomer, and exposure of the hybridization site for thesecond haplomer. After duplex formation with the second FKM-haplomer(see, FIG. 26 ), the preparations of mFKBP fusions of sfGFP and RenillaN- and C-terminal fragments (as above) are added. This allows the secondstage of the LD-TAPER process to proceed, with FKM-mFKBP binding, andthereby juxtaposing the polypeptide split-protein pair for maturefolding (see FIG. 27 ).

The sfGFP signal is fluorescence at the same emission maximum as forfluorescein, and is monitored by means of a spectrophotometer withfluorescent reading facility (Tecan). The enzymatic activity of Renillaluciferase is assessed by means of commercial kits for this enzyme(Promega), using coelenterazine substrate, and purified Renillaluciferase (RayBiotech) as a positive control. Luminescence isquantified by means of a standard luminometer (Berthold).

In a dose-response experimental design, equimolar amounts of sfGFP N-and C-terminal mFKBP-fusions are mixed with first-stage FKM-haplomers“unlocked” in the presence of specific target template as above. Ratiosof the N- and C-terminal mFKBP fusions to FKM-haplomer range from 1:1 to10:1, Analogous experiments are established with Renilla N- andC-terminal mFKBP fusions. After a 16 hour incubation at 25° C., reportersignals are assayed as appropriate for both sfGFP and Renillaluciferase.

Since homoligand LB-TAPER for split-protein folding has an efficiencylimitation if the haplomer/template hybridizations are performed as thefirst step (as shown with Equations 4.1, 4.2), in addition toexperimental approach “A” a second (“B”) protocol is also followed, inaccord with Equations 5.1 and 5.2. Here the locked-TAPER first bottlehaplomer bearing its FKM tag (see, FIG. 26 ) is hybridized to templateas before, but then treated with the N-terminal sfGFP or RenillaC-terminal mFKBP fusions. In a separate reaction, the secondFKM-haplomer is treated with the C-terminal sfGFP or Renilla N-terminalmFKBP fusions. After 2 hours at 25° C., each pre-assembledFKM-haplomer/template/mFKBP-polypeptide is mixed in equimolar amounts.After a 16 hour incubation at 25° C., reporter signals are assayed asappropriate for both sfGFP and Renilla luciferase. During the incubationtime, mature protein folding is enable. Note that long serine-glycinelinkers are used to accommodate the orientation of the mFKBP domainbinding to its cognate FKM monovalent ligand (see, FIG. 27 ).

For both experimental designs “A” and “B”, comparable time-courseexperiments are also performed after the two-stage assembly of allLD-TAPER components has been completed. In protocol “A”, thiscorresponds to the initial hybridizations for both FKM-haplomers;followed by the addition of the split-protein mFKBP-polypeptides; forprotocol “B”, this corresponds to the separate pre-assembly ofFKM-haplomers with specific split-protein mFKBP-polypeptides, followedby combination of the two haplomeric complexes. For each arrangement,assayable samples taken at a series of time points: 15, 30, 45, and 60minutes; and 1, 2, 4, 6, 8, and 16 hours.

Specificity of the template-mediated LB-TAPER assembly may bedemonstrated by the use of a blocking oligonucleotide that correspondsto the same sequence as the second haplomer, but lacking any appendedtag. A molar excess of such an oligonucleotide effectively inhibits theassembly reaction, whereas the assembly process is unaffected by excessoligonucleotide of the same length but with scrambled sequence.

Example 4: Improved Responses with Cysteine Mutants of FKBP and FRBDimerization Domains with Gaussia Luciferase Split-Protein Assays

This example describes the application of cysteine mutants of FKBP andFRB dimerization domains, C225 and C61S, respectively, in split-proteinassays with Gaussia luciferase. In this case, the homodimerization agentAP20187 (Clontech Labs) was used to dimerize N-terminal Gaussiasplit-protein fragment-FKBP and FKBP-C-terminal Gaussia split-proteinfragment fusions, and thus greatly accelerate Gaussia split-proteinassembly. In a likewise fashion, the heterodimerization agent rapamycin(Sigma) was used to dimerize N-terminal Gaussia split-proteinfragment-FKBP and FRB-C-terminal Gaussia split-protein fragment fusions,with a concomitant Gaussia assembly rate increase. For the FKBP domain,the C22S cysteine mutation was introduced in the context of theadditional F36V mutation, as described above.

Protein fragments with and without the cysteine mutations were expressedin E. coil in standard fashion, such that their purifications could beeffected by means of hexhistidine affinity tags, on solid-phaseimmobilized metal affinity chromatography (IMAC) beads (IMAC-Dynabeads,Thermofisher Scientific). Proteins purified inn this manner werevisualized on 16% Tricine gels (FIG. 28 ).

The sequence of the N-terminal Gaussia split-protein fragment-FKBPsingle F36V mutant fusion protein is:MKPTENNEDFNIVAVASNFATTDLDADRGKLPGKKLPLEVLKEMEANARKAGCTRGCLICLSHIKCTPKIVIKKFIPGRCHTYEGDKESAQGGIG-GSGGGGSSGGG-GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGHPPHATLVFDVELLKLEGGSG-HHHHHH (SEQID NO:47), where hyphens demarcate (in order) the N-terminal Gaussiasegment, a serine-glycine linker segment, the C-terminal FKBP F36Vmutant, and the hexahistidine tag, with the mutant valine residue (F36V)in bold.

The sequence of the N-terminal Gaussia split-protein fragment-FKBPC/F36V double mutant fusion protein is likewise:MKPTENNEDFNIVAVASNFATTDLDADRGKLPGKKLPLEVL.KEMEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESAQGGIG-GSGGGGSSGGG-GVQVETISPGDGRTFPKRGQTSVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGIIPGIIPPIIATLVFDVELLKLEGGSG-HHHHHH (SEQ D NO:48), where the mutant valine (F36V) residue isin bold, and mutant serine (C22S) residue is underlined.

The sequence of the FKBP-C-terminal Gaussia split-protein fragmentsingle FKBP F36V mutant fusion protein is:MHHHHHH-GGSG-GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE-GSGGGGSSGGG-EAIVDIPEIPGFKDLEMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQRCATFASKIQGQVDKIKGAGGD (SEQ ID NO:49),where hyphens demarcate (in order) the hexahistidine tag, a short serineglycine linker, the N-terminal FKBP segment, a longer serine-glycinelinker segment, and the C-terminal Gaussia sequence, with the mutantFKBP valine residue (F36V) in bold.

The sequence of the FKBP-C-terminal Gaussia split-protein fragmentdouble FKBP C22S/F36V mutant fusion protein is likewise:MHHHHHH-GGSG-GVQVETISPGDGRTFPKRGQTSVVHYTGMLEDGKKVDSSRDRNKPEKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE-GSGGGGSSGGG-EAIVDIPEIPGFKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQRCATFASKIQGQVDKIKGAG GD (SEQ IDNO:50), where the FKBP mutant valine (F36V) residue is in bold, andmutant serine (C225) residue is underlined.

The sequence of the wild-type FRB-C-terminal Gaussia split-proteinfragment mutant C61S mutant fusion protein is:MHHHHHH-GGSG-EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ-GSGGGGSSGG-GEAIVDIPEIPGFKDLEPMEQFIAQVDLCVDCYYGCLKGLANVQCSDLLKKWLPQRCATFASKIQGQVDKIKGAGGD (SEQ ID NO:51), where hyphensdemarcate (in order) the hexahistidine tag, a short serine glycinelinker, the N-terminal FRB segment, a longer serine-glycine linkersegment, and the C-terminal Gaussia sequence.

The sequence of the C61S mutant FRB-C-terminal Gaussia split-proteinfragment fusion protein is likewise:MHHHHHH-GGSG-EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWSRKYMKSGNVKDLTQAWDLYYFIVFRRISKQ-GSGGGGSSGG-GEAIVDIPEIPGFKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQRCATFASKIQGQVDKIKGAGGD (SEQ ID NO:52), where the mutantserine (C61S) residue is underlined.

After expression and elution from MAC magnetic beads with imidazole,proteins were assayed (Bradford reagent) and visualized on 16% Tricinegels (Thermofisher).

Assays were set up in 20 μl volumes with protein fragments (single or inpaired mixes) at a final concentration 0.3 μM, with or without AP20187or rapamycin as appropriate (both at 0.15 μM final). At suitabletime-points, 2 μl samples were taken and assayed for luminescence in aBerthold luminometer, in tubes with coelenterazine (2.5 μM final) in 50μl PBS with 5 mM sodium bromide. Representative data are shown in FIG.29 . Co-incubated preparations of N-terminal Gaussia segment-FKBP-F36Vfusion and FKBP-F36V-C-terminal Gaussia segment fusion showed enhancedresponses in the presence of the homodimerizer AP20187 (FIG. 29 , Panel1), while greater equivalent responses were seen with the use ofcorresponding fusions with the FKBP-F36V-C225 double mutations (FIG. 29, Panel 2). Co-incubated preparations of N-terminal Gaussiasegment-FKBP-F36V fusion and wild-type FRB-C-terminal Gaussia segmentfusion showed marked enhancement with the heterodimerizer rapamycin(FIG. 29 , Panel 3), which was further promoted in the context of theFKBP-F36V-C225 double mutation in combination with e FRB 0615 mutation(FIG. 29 , Panel 4). Results demonstrated both the efficacy ofdimerizers in this context, and functionality of the FKBP and FRBcysteine-serine mutations.

Example 5: Demonstration of Oligonucleotide Conjugate Formation with theMFI2 Monovalent FKBP Ligand

This example describes the derivatization of thiol-labeledoligonucleotides specifically with MFL2, the monovalent ligand for theF36V mutant form of FKBP. MFL2 was solubilized in DMSO at aconcentration of 100 mM. To prepare for conjugation, 5 nmol ofoligonucleotides modified at their 5′- or 3′-ends with disulfidemoieties (IDT Corp.) were treated with 1 mM tris-carboxyethylphosphine(TCEP) for 12-16 hours in 50 μl PBS to reduce the disulfides and producefree thiol groups. Following this, the reduced oligonucleotides weredesalted on Micro-Biospin P6 columns (Bio-Rad) into 10 mM Tris pH 7.4,and then used immediately for conjugation. Conjugation reactions wereperformed in 100 μl volumes, in 50 mM phosphate buffer pH 7.0/100 mMNaCl, with 4 nmol of reduced thiol-oligonucleotides, 2 mM TCEP and 400nmol MFL2, all with al concentration of 50% DMSO. The 100-fold molarexcess of MFL2 was used to drive the reaction between the MFL2 maleimidomoiety and the thiol group on the relevant oligonucleotides towardscompletion; the indicated DMSO level was necessary to maintainsolubility of the hydrophobic MFL2 molecule at the desiredconcentration. Reactions were allowed to proceed for 12-16 hours at roomtemperature, and then free MFL2 was removed from the preparations by twosuccessive rounds of buffer exchange through Micro-Biospin P6 columnsequilibrated with PBS. Conjugate preparations were compared withcorresponding unmodified thiol-oligonucleotides on denaturing 15% ureagels. It was apparent that under the conditions used, the conjugationreaction progressed essentially towards completion (FIG. 30 ).

The conjugates produced in this Example were: #407-MFL2 (3′ conjugation)GTCCAG ATGTCTTTGC-MFL2 (SEQ ID NO:44); #408-MFL2 (3′ conjugation)GCTGTGTCCTGAAG AAA-MFL2 (SEQ ID NO:45); #409-MFL2 (5′ conjugation)MFL2-TTTCTTCAGGACACA GC (SEQ ID NO:46),

Example 6: Use of Oligonucleotide Conjugates with Mutant FKBP-BindingMonovalent Compounds in LD-TAPER for split-protein LD-TAPER

In this Example, LD-TAPER is demonstrated both with Architecture 1, andtwo approaches to demonstrating it with Architecture 2.

Architecture

In this case, mutually complementary oligonucleotides bearing 5′ (#409)or 3′ (#408) thiol modifications were reacted with the compound MFL2(Example 5), and desalted/buffer exchanged into PBS by two rounds ofpassage through Micro-Biospin P6 columns (Bio-Rad) previouslyPBS-equilibrated. Resulting conjugates were then incubated for 1 hour atroom temperature with purified Gaussia split-protein segments fused withthe FKBP-F36V-C22S double mutant domain (FIG. 28 ), with thecorresponding unconjugated oligos used as controls. The N-terminalGaussia-FKBP-F36V-C225 fusion (code (C) in this Example) was thusincubated with conjugate #408-MFL2 (Example 5) or control #408 alone,while the FKBP-F36V-C225-C-terminal Gaussia fusion (code (D) in thisExample) was incubated with conjugate #409-MFL2 (Example 5) or control#409 alone. Each preparation (15 pl PBS final) contained 60 pmol ofprotein fragments, and 30 pmol of conjugate or oligonucleotide alone.The interaction products of FKBP domain protein fusions and MFL2conjugates constitute a particular class of LD-TAPER haplomers.Following this, 5 μl samples of these pre-incubations were used inisolation or as appropriate mixtures, with volumes adjusted to a final20 μl. At suitable time-points, 2 μl samples were then taken and assayedfor luminescence in a Berthold luminometer, in tubes with coelenterazine(2.5 μM final) in 50 μl PBS with 5 mM sodium bromide. Data for threetime points are shown in FIG. 31 . Markedly increased luminescence overtime was observed where the mutually complementary LD-TAPER haplomers(C)-#408MFL2 and (D)-#409MFL2 were co-incubated together, but no sucheffect was seen with equivalent co-incubations with the unconjugatedoligos alone, nor any haplomer in isolation (FIG. 31 ).

Architecture 2—Solid-Phase Capture

In this strategy, a template strand bearing a 5′ desthiobiotinmodification was used. Gaussia-FKBP LD-TAPER haplomers (complexesbetween Gaussia luciferase-FKBP fragments and MFL2-modifiedoligonucleotides) can hybridize in mutual spatial proximity onto thistemplate, enabling Gaussia protein folding assembly and full activitygeneration. This process is depicted in FIG. 32 , and corresponds toArchitecture 2 (FIG. 10 ). Control unmodified oligonucleotides can alsohybridize to the same template, but cannot spatially force thejuxtaposition of the Gaussia fragments in the same manner. Thisprocedure has the advantage that background luminescence fromspontaneous Gaussia self-assembly is substantially reduced. Followingthe hybridization step, the templates and all co-hybridized moleculesare bound to streptavidin magnetic beads, and all other solutioncomponents washed away. After this, elutions from the magnetic beads areeffected with free D-biotin, which displaces the streptavidin-associateddesthiobiotin-tagged template complexes, by virtue of its much higherbinding affinity. Gaussia luciferase assays are then performed on eacheluted preparation. Specifically, in a volume of 18 μl PBS, 120 pmol ofpurified Gaussia protein-FKBP fusion fragments were incubated for 1 hourat room temperature with either 60 pmol oligonucleotide-MFL2 complexes(FIG. 30 ) or corresponding unmodified thiol-oligonucleotides. Followingthis, 7.5 μl of these preincubation reactions were added to finalreactions of 25 μl PBS, either alone or in combination. In this Example,the following preparations were used:

N-terminal Gaussia fragment-FKBP-F36V (code=(A))

FKBP-F36V-C-terminal Gaussia fragment (code=(B))

N-terminal Gaussia fragment-FKBP-F36V-C22S (code=(C))

FKBP-F36V-C225-C-terminal Gaussia fragment (code=(D))

With the above code and the oligonucleotide descriptions of Example 5,the LD-haplomer preparations are:

(A)-#407-MFL2

(B)-#409-MFL2

(C)-#407-MFL2

(D)-#409-MFL2

Control preparations used the same Gaussia-FKBP fusion fragmentsincubated with corresponding unconjugated oligonucleotides.

Appropriate protein fragment/conjugate/control oligonucleotidepreparations were incubated for 30 minutes at room temperature inn thepresence of 25 pmol of template oligonucleotide (#230) bearing a 5′desthiobiotin moiety: Desthiobiotin-AGCTGTGTCCTGAAGAAAGCAAAGACATCTGG.ACA (SEQ ID NO:53).

Following this, 25 μl (100 μg) of hydrophilic streptavidin magneticbeads (New England Biolabs, pre-washed twice in 1 ml PBS and resuspendedin the original volume of PBS) were added to each tube, and incubatedfor a further 1 hour at room temperature. The beads were thenmagnetically separated from the supernatants, and washed ×1 with 0.5 mlof PBS. The magnetic bead pellets in each tube were resuspended in 50 μlof PBS, and free D-biotin was added to 10 mM for a 30 minute roomtemperature incubation. Supernatants were then taken by magneticseparation, and 2 μl samples of each assayed for Gaussia luciferaseactivity as for Example 6. Results are shown in FIG. 33 , where markedlyelevated luminescence readings were obtained only with combinedN-terminal and C-terminal Gaussia oligo-MFL2 conjugate complexes(LD-TAPER haplomers), for both the F36V and F36V-C225 forms of FKBP. Nosuch responses were seen when the MFL2-modified oligonucleotides werereplaced with corresponding unmodified oligonucleotides (FIG. 33 ). Thisdata is thus consistent with templated assembly of Gaussia fragmentsmediated via LD-TAPER in an Architecture 2 templating system.

Architecture 2—Solution Phase

The utility of LD-TAPER mediated via an Architecture 2 templating systemin solution was also investigated. Preincubations were set up in thesame manner as for the above Architecture 2—Solid-phase Capture Example,for the Gaussia fragments fused with the FKBP-F36V-C22S double mutantdomain.

In this case, after the initial preincubations, combinations of LD-TAPERhaplomers or controls replacing the MFL2-oligonucleotide conjugates withunmodified thiol oligonucleotides were simply mixed with either aspecific template or a scrambled oligonucleotide with the same lengthand base composition as the template. The specific template wasidentical to the template oligonucleotide #230 above, except without the5′-desthiobiotin modification. The scrambled control oligo was:GACTAGACGGCCAGGGAGACGAATACATATTCAAT (SEQ ID NO:54)

Samples (2 μl) of these preparations were then assayed at certain timeintervals for Gaussia luciferase activity, in the same manner as forExample 6. Results (FIG. 34 ) at 2 hour post-initiation show that asubstantial increase in the luminescent signal was observed for specifictemplate over the scrambled control (˜2.5-fold increase), againconsistent with template-mediated assembly of Gaussia luciferase bymeans of LD-TAPER haplomers. The nature of the templating process thusconforms to that depicted in FIG. 32 , except for the absence of thedesthiobiotin moiety.

Various modifications of the described subject matter, in addition tothose described herein, will be apparent to those skilled in the artfrom the foregoing description. Such modifications are also intended tofall within the scope of the appended claims. Each reference (including,but not limited to, journal articles, U.S. and non-U.S. patents, patentapplication publications, international patent application publications,gene bank accession numbers, and the like) cited in the presentapplication is incorporated herein by reference in its entirety.

What is claimed is:
 1. A haplomer-ligand complex comprising: a haplomer,wherein the haplomer comprises a polynucleotide that is substantiallycomplementary to a target nucleic acid molecule; and a ligand linked tothe 5′ or 3′ terminus of the haplomer, wherein the ligand comprises aligand partner binding site.
 2. A composition or kit comprising a firsthaplomer-ligand complex of claim 1 and a second haplomer-ligand complexof claim 1, wherein: the ligand of the first haplomer-ligand complex islinked to the 5′ terminus of the polynucleotide of the firsthaplomer-ligand complex; and the ligand of the second haplomer-ligandcomplex as linked to the 3′ terminus of the polynucleotide of the secondhaplomer-ligand complex.
 3. A bottle haplomer-ligand complex comprising:a) a bottle haplomer wherein the bottle haplomer comprises apolynucleotide, wherein the polynucleotide comprises: i) a first stemportion comprising from about 10 to about 20 nucleotide bases; ii) ananti-target loop portion comprising from about 16 to about 40 nucleotidebases and having a first end to which the first stein portion is linked,wherein the anti-target loop portion is substantially complementary to atarget nucleic acid molecule; and iii) a second stem portion comprisingfrom about 10 to about 20 nucleotide bases linked to a second end of theanti-target loop portion, wherein the first stem portion issubstantially complementary to the second stem portion; and b) a ligandlinked to the terminal end of either the first stem portion or thesecond stem portion, wherein the ligand comprises a ligand partnerbinding site; wherein the T_(m) of the anti-target loop portion:targetnucleic acid molecule is greater than the T_(m) of the first stemportion:second stem portion.
 4. A composition or kit comprising: abottle haplomer-ligand complex of claim 3; and a second haplomer-ligandcomplex comprising: a nucleotide portion comprising from about 6 toabout 20 nucleotide bases that is substantially complementary to thestem portion of the bottle haplomer-ligand complex that is linked to theligand of the bottle haplomer-ligand complex; and a ligand linked to the5′ or 3′ terminus of the nucleotide portion of the secondhaplomer-ligand complex, wherein the ligand comprises a ligand partnerbinding site; wherein the T_(m) of the second haplomer-ligandcomplex:first or second stem portion linked to the ligand of the bottlehaplomer-ligand complex is less than or equal to the T_(m) of the firststem portion:second stem portion of the bottle haplomer-ligand complex.5. A compound having the formula:

where m is from 3 to 6; having the formula:

where m is from 3 to 6; having the formula:

where n is from 1 to 6; having the formula:

where x is from 1 to 6; having the formula:

where x is from 1 to 6; or having the formula:


6. A fusion protein comprising a fragment of a protein of interest fusedto a ligand binding domain, wherein the ligand binding domain is an FKBPdomain or an FRB domain.
 7. A composition or kit comprising a firstfusion protein of claim 6 and a second fusion protein of claim 6,wherein the protein of interest of the first fusion protein and theprotein of interest of the second fusion protein can dimerize or foldtogether.
 8. A method for the directed assembly of a protein comprising:contacting a target nucleic acid molecule with a first haplomer-ligandcomplex of claim 1; contacting the target nucleic acid with a secondhaplomer-ligand complex of claim 1; contacting the first haplomer-ligandcomplex with a first fusion protein of claim 6; and contacting thesecond haplomer-ligand complex with a second fusion protein of claim 6;wherein the ligand of ate first haplomer-ligand complex is linked to the5′ terminus of the polynucleotide of the first haplomer-ligand complex;wherein the ligand of the second haplomer-ligand complex is linked tothe 3 terminus of the polynucleotide of the second haplomer-ligandcomplex; wherein the polynucleotide of the first haplomer-ligand complexis substantially complementary to a target nucleic acid molecule;wherein the polynucleotide of the second haplomer-ligand complex issubstantially complementary to the target nucleic acid molecule at asite in spatial proximity to the polynucleotide of the firsthaplomer-ligand complex; wherein the ligand of the first haplomer-ligandcomplex and the ligand binding domain of the first fusion protein caninteract; and wherein the ligand of the second e ligand binding domainof the second fusion protein can interact; thereby resulting in thefolding or dimerization of the fragment of the protein of interest ofthe first fusion protein with the fragment of the protein of interest ofthe second fusion protein.
 9. A method for the directed assembly of aprotein comprising: contacting a target nucleic acid molecule with afirst haplomer-ligand complex of claim 1; contacting the target nucleicacid with haplomer-ligand complex of claim 1; contacting the firsthaplomer-ligand complex with a first fusion protein of claim 6; andcontacting the second haplomer-ligand complex with a second fusionprotein of claim 6; wherein the ligand of the first haplomer-ligandcomplex is finked to the 5′ terminus of the polynucleotide of the firsthaplomer-ligand complex; wherein the ligand of the secondhaplomer-ligand complex is linked to the 3′ terminus of thepolynucleotide of the second haplomer-ligand complex; wherein thepolynucleotide of the first haplomer-ligand complex is substantiallycomplementary to a target nucleic acid molecule; wherein thepolynucleotide of the second haplomer-ligand complex is substantiallycomplementary to the target nucleic acid molecule at a site in spatialproximity to the polynucleotide of the first haplomer-ligand complex;wherein the ligand of the first haplomer-ligand complex and the ligandbinding domain of the first fusion protein can interact; and wherein theligand of the second haplomer-ligand complex and the ligand bindingdomain of the second fusion protein can interact; thereby resulting inthe folding or dimerization of the fragment of the protein of interestof the first fusion protein with the fragment of the protein of interestof the second fusion protein.
 10. A method for the directed assembly ofa protein comprising: contacting a target nucleic acid molecule with acomplex formed by the interaction of a first haplomer-ligand complex ofclaim 1 with a first fusion protein of claim 6, wherein the ligand ofthe first haplomer-ligand complex is linked to the 5′ terminus of thepolynucleotide of the first haplomer-ligand complex, and wherein theligand of the first haplomer-ligand complex interacts with the ligandbinding domain of the first fusion protein; and contacting the targetnucleic acid molecule with a complex formed by the interaction of asecond haplomer-ligand complex of claim 1 with a second fusion proteinof claim 6, wherein the ligand of the second haplomer-ligand complex islinked to the 5′ terminus of the polynucleotide of the secondhaplomer-ligand complex, and wherein the ligand of the secondhaplomer-ligand complex interacts with the ligand binding domain of thesecond fusion protein; thereby resulting in the folding or dimerizationof the fragment of the protein of interest of the first fusion proteinwith the fragment of the protein of interest of the second fusionprotein.
 11. A method for the directed assembly of a protein comprising:contacting a target nucleic acid molecule with a complex formed by theinteraction of a first haplomer-ligand complex of claim 1 with a firstfusion protein of claim 6, wherein the ligand of the firsthaplomer-ligand complex is linked to the 5′ terminus of thepolynucleotide of the first haplomer-ligand complex, and wherein theligand of the first haplomer-ligand complex interacts with the ligandbinding domain of the first fusion protein; and contacting the targetnucleic acid molecule with a complex formed by the interaction of asecond haplomer-ligand complex of claim 1 with a second fusion proteinof claim 6, wherein the ligand of the second haplomer-ligand complex islinked to the 5′ terminus of the polynucleotide of the secondhaplomer-ligand complex, and wherein the ligand of the secondhaplomer-ligand complex interacts with the ligand binding domain of thesecond fusion protein; thereby resulting in the folding or dimerizationof the fragment of the protein of interest of the first fusion proteinwith the fragment of the protein of interest of the second fusionprotein.
 12. A method for he directed assembly of a protein comprising:contacting a target nucleic acid molecule with a bottle haplomer-ligandcomplex of claim 3; contacting the target nucleic acid with a secondhaplomer-ligand complex of claim 1, wherein the second haplomer-ligandcomplex comprises a nucleotide portion that is substantiallycomplementary to the stem portion of the bottle haplomer-ligand complexthat is linked to the ligand of the bottle haplomer-ligand complex;contacting the bottle haplomer-ligand complex with a first fusionprotein of claim 6, wherein the ligand of the bottle haplomer-ligandcomplex and the ligand binding domain of the first fusion protein caninteract; and contacting the second haplomer-ligand complex with asecond fusion protein of claim 6, wherein the ligand of the secondhaplomer-ligand complex and the ligand binding domain of the secondfusion protein can interact; thereby resulting in the folding ordimerization of the fragment of the protein of interest of the firstfusion protein with the fragment of the protein of interest of thesecond fusion protein.
 13. A method for the directed assembly of aprotein comprising: contacting a target nucleic acid molecule with abottle haplomer-ligand complex of claim 3; contacting the target nucleicacid with a second haplomer-ligand complex of claim 1, wherein thesecond haplomer-ligand complex comprises a nucleotide portion that issubstantially complementary to the stem portion of the bottlehaplomer-ligand complex that is linked to the ligand of the bottlehaplomer-ligand complex; contacting the bottle haplomer-ligand complexwith a first fusion protein of claim 6, wherein the ligand of the bottlehaplomer-ligand complex and the ligand binding domain of the firstfusion protein can interact; and contacting the second haplomer-ligandcomplex with a second fusion protein of claim 6, wherein the ligand ofthe second haplomer-ligand complex and the ligand binding domain of thesecond fusion protein can interact; thereby resulting in the folding ordimerization of the fragment of the protein of interest of the firstfusion protein with the fragment of the protein of interest of thesecond fusion protein.
 14. A method for the directed assembly of aprotein comprising: contacting a target nucleic acid molecule with abottle haplomer-ligand complex of claim 3; contacting the target nucleicacid molecule with a second haplomer-ligand complex of claim 1, whereinthe second haplomer-ligand complex comprises a nucleotide portion thatis substantially complementary to the stem portion of the bottlehaplomer-ligand complex that is linked to the ligand of the bottlehaplomer-ligand complex; contacting the bottle haplomer-ligand complexwith a first fusion protein of claim 6, wherein the ligand of the bottlehaplomer-ligand complex and the ligand binding domain of the firstfusion protein can interact; and contacting the second haplomer-ligandcomplex with a second fusion protein of claim 6, wherein the ligand ofthe second haplomer-ligand and complex and the ligand binding domain ofthe second fusion protein can interact; thereby resulting in the foldingor dimerization of the fragment of the protein of interest of the firstfusion protein with the fragment of the protein of interest of thesecond fusion protein.
 15. A method for the directed assembly of aprotein comprising: contacting a target nucleic acid molecule with abottle haplomer-ligand complex of claim 3; contacting the target nucleicacid molecule with a second haplomer-ligand complex of claim 1, whereinthe second haplomer-ligand complex comprises a nucleotide portion thatis substantially complementary to the stem portion of the bottlehaplomer-ligand complex that is linked to the ligand of the bottlehaplomer-ligand complex; contacting the bottle haplomer-ligand complexwith a first fusion protein of claim 6, wherein the ligand of the bottlehaplomer-ligand complex and the ligand binding domain of the firstfusion protein can interact; and contacting the second haplomer-ligandcomplex with a second fusion protein of claim 6, wherein the ligand ofthe second haplomer-ligand complex and the ligand binding domain of thesecond fusion protein can interact; thereby resulting in the folding ordimerization of the fragment of the protein of interest of the firstfusion protein with the fragment of the protein of interest of thesecond fusion protein.